results and discussion 3. purification of rice bran lipase,...
TRANSCRIPT
RESULTS AND DISCUSSION
3. Purification of Rice Bran Lipase, Molecular
Cloning and Expression in E. coli
Purification and heterologous expression of RBL
67
In plants, most of the studies on lipases have been directed towards isolation
and characterization of these enzymes with view to understand their biological role. In
contrast, remarkably little is known about the molecular cloning and functional
expression of plant lipases, despite their fundamental importance. Rice bran is known
to contain two soluble lipases, Lipase-I and Lipase-II which have been purified and
characterized (Funatsu et al., 1971; Aizono et al., 1973; Aizono et al., 1976) and a
phospholipase (Bhardwaj et al., 2001). Lipase-II the major lipase of rice bran is
hitherto referred to as RBL. The results on the cloning and expression of RBL in E.
coli is presented and discussed. RBL was purified to homogeneity and its NH2-
terminal determined to abet in the cloning.
Purification of RBL
The lyophilized concentrate of the rice bran crude extract was dialyzed against
50 mM sodium phosphate buffer pH 7.4 and applied to a DEAE Sepharose ion
exchange column pre-equilibrated in 50 mM sodium phosphate buffer, pH 7.4. The
column was washed with equilibration buffer to remove unbound protein until the
A280 was zero. The bound protein was selectively eluted with 50 mM sodium
phosphate buffer pH 7.4 containing 0.2 M KCl (Figure 3. 1). The fractions showing
lipolytic activity were pooled and subjected to 80 % (NH4)2SO4 precipitation. This
fraction was further purified by size exclusion chromatography using a Superdex-75
(10 mm 30 cm) column. The concentrate was loaded to a Superdex-75 column pre-
equilibrated with five column volumes of 50 mM sodium phosphate buffer, pH 7.4.
The fractions showing pNPA hydrolytic activity were pooled (Figure 3. 2) and stored
at 4 C for further studies. The purified RBL had a specific activity of 189.7 ± 7.9
U/mg protein. The purification is summarized in Table 3. 1.
Table 3. 1. Summary of RBL purification from defatted rice bran.
Purification step
Total
Protein
(mg)
Total
Activity
(U)
Specific
Activity
(U/mg)
Fold
purification
Yield
(%)
Crude extract 147.5 1425 9.7 ± 1.7 - 100
DEAE Sepharose
chromatography 19.7 744 84.8 ± 3.3 8.8 52.2
Superdex-75
chromatography 2.0 387 189.7 ± 7.9 19.6 27.2
These are the results of a typical purification starting from 10 g of defatted rice bran powder. These values are reproduced in
three separate purifications.
Purification and heterologous expression of RBL
68
0 50 100 150 200
0.2
0.4
0.6
0.8
1.0
1.2
0.2
M K
Cl
Elution volume (mL)
A2
80
nm
0.000
0.025
0.050
0.075
0.100
0.125
Ac
tivity
(Un
its/m
L)
0 3 6 9 12 15 18 21 24 27
75
150
225
300
375
Elution volume (mL)
A2
80
nm
(m
AU
)
0.07
0.14
0.21
0.28
0.35
Ac
tivity
(Un
its/m
L)
Figure 3. 1. DEAE Sepharose chromatography elution profile of RBL. The active
fractions pooled are indicated (←→). Protein ( ̶ • ̶ ) and pNPA hydrolytic activity (▪).
.
Figure 3. 2. Superdex-75 10/30 HR chromatography elution profile of RBL. The arrow
shows the active fractions that were pooled. Protein ( ̶ • ̶ ) and pNPA hydrolytic activity
(▪).
Purification and heterologous expression of RBL
69
A B
Figure 3. 3. Native-PAGE (10 % T, 2.7 % C) profile of purified RBL. The gel was stained
for A) protein and B) lipase activity. Arrow indicates RBL.
Evaluation of activity and homogeneity of RBL
The homogeneity of purified RBL was determined by Native-PAGE (10 % T,
2.7 % C), SDS-PAGE (12.5 % T, 2.7 % C) and RP-HPLC. Purified RBL was
subjected to 10 % Native-PAGE (10 % T, 2.7 % C) and located by protein and lipase
activity staining. A single species was observed both by protein staining using CBB
R-250 and specific enzyme staining with 1-napthyl acetate and tetrazotized-o-
dianisidine dye (Figure 3. 3). The relative mobility of the protein stained for lipase
activity was identical to the protein band detected by Native-PAGE (10 % T, 2.7 %
C).
The purified protein was separated on 12.5 % SDS-PAGE (12.5 % T, 2.7 % C)
and visualized by CBB R-250 staining (Figure 3. 4A). A plot of log molecular weight
versus migration rate of the standard marker proteins showed a R2 = 0.986 (Figure 3.
4B). The molecular mass of RBL was 34,000 ± 1,530 Da (Figure 3. 4). The exact
molecular weight as determined by ESI-MS showed a mass of 35,293 Da (Figure 3.
5). RBL resolved as a single peak by RP-HPLC on a Discovery C18 column (4.6 250
mm, 5 µm) using a binary gradient of 70 % acetonitrile containing 0.05 % TFA and
water containing 0.1 % TFA (Figure 3. 6). All these results reckon the apparent
homogeneity of purified RBL.
Purification and heterologous expression of RBL
70
B
2 3 4 5 6
1.2
1.4
1.6
1.8
y = -0.138x + 2.075
R² = 0.986
Lo
g M
r
Migration rate (cm)
RBL
29
43
6.5
14.3
20.1
kDa M 1
A
0
%
100 35293.00
35218.00
35149.00
34959.00
3509334460.00
34570.0034817.00
35365.00
35437.00
35561.00
35706.0035774.00 36020.00
TOF MS ES+
1.11e5
34400 34600 34800 35000 35200 35400 35600 35800 36000mass
Figure 3. 4. Molecular weight determination by SDS-PAGE. A). SDS-PAGE (12.5 % T,
2.7 % C) profile of purified RBL. Lane M, Molecular weight markers: ovalbumin (43,000
Da), carbonic anhydrase (29,000 Da) and soybean trypsin inhibitor (20,000 Da), lysozyme
(14,300 Da), aprotinin (6,500 Da) and Lane 1, RBL. B) A plot of relative mobility vs log
molecular weight.
Figure 3. 5. ESI mass spectrum of purified RBL.
Purification and heterologous expression of RBL
71
0 2 4 6 8 10 12
2
4
6
8
10
pI
Distance from the cathode (cm)
Pepsinogen
Amyloglycosidase
Glucose oxidaseSoybean trypsin inhibitor
b-lactoglobinBovine carbonic anhydrase B
Human carbonic anhydrase B
Horse myoglobin band acidic Horse myoglobin band basic
Lentil lectin-acidic band Lentil lectin-middle band
Lentil lectin-basic band
trypsinogen
RBL
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0
min
-20
-10
0
10
20
30
40
50
60
70
80
90
mA
UCh2-280nm,4nm (1.00)
Ch1-230nm,4nm (1.00)
29.2
10
Figure 3. 6. RP-HPLC profile indicating the homogeneity of the purified RBL. The
protein was analyzed using a Discovery C18 column (4.6 250 mm, 5 µm) on a Waters
Associate HPLC, using a linear gradient of 0.1 % TFA and 70 % acetonitrile containing 0.05
% TFA.
Figure 3. 7. Determination of isoelectric point of RBL. The plot shows distance moved
from cathode vs pI. Standard pI markers used are shown in the figure.
Purification and heterologous expression of RBL
72
Triacylglycerol lipase (Pubmed Acc. No. BAC 83592.1)
MERWRCVSVLALVLLLSNASHGRDISVQHSQQTLNYSHTLAMTLVEYASAVYMTDLTA
LYTWTCSRCNDLTQGFEMKSLIVDVENCLQAFVGVDYNLNSIIVAIRGTQENSMQNWI
KDLIWKQLDLSYPNMPNAKVHSGFFSSYNNTILRLAITSAVHKARQSYGDINVIVTGH
SMGGAMASFCALDLAINLGSNSVQLMTFGQPRVGNAAFASYFAKYVPNTIRVTHGHDI
VPHLPPYFSFLPHLTYHHFPREVWVNDSEGDITEQICDDSGEDPNCCRCISTWSLSVQ
DHFTYLGVDMEADDWSTCRIITAENVRQLQKDLASNIIVSKHSVDVTIVEPSSQTY
Isoelectric focusing (IEF) using pre-casted ampholine PAG, pH range 3-10 was
carried out with standard pI markers in the pH range of 3.5-9.5. The purified RBL
showed a pI of 9.14, which is close to the pI reported for Lipase-II (Aizono et al.,
1976) (Figure 3. 7). The purified protein was electro blotted on a PVDF membrane
using semidry blotting unit. The protein was subjected to NH2-terminal sequence
analysis by Edman degradation. The NH2-terminal sequence ASHGRDISVQH----
was obtained. BLAST search of this sequence showed similarity to a putative TAG
lipase of Oryza sativa, GenBank Acc. No. BAC83592.1 (Figure 3. 8).
Prediction of antigenic peptides in RBL
Previous attempts in our laboratory to raise antibodies to RBL were futile.
Therefore it was proposed to use a predicted antigenic peptide to raise antibodies
against RBL. The plot of antigenicity along the polypeptide chain of RBL as predicted
by the algorithm of Hopp and Woods (Hopp & Woods, 1981) (Figure 3. 9) was used.
The plot displays the variation of the antigenic index as a function of amino acid
position. The higher the antigenic index, the more likely would be that antibodies
would “see” those groups of residues. The antigenic peptides determined by Antigen
Prediction tool from Priceton BioMolecules Corporation (PA, USA) are listed in
Table 3. 2 in the order of their antigenic potential. Among the three peptides, the
sequence of peptide No. 1 was used.
Figure 3. 8. Protein sequence retrieved from the databank. The NH2-terminal obtained
from Edman degradation of the purified RBL is highlighted as bold and underlined.
Table 3. 2. Sequence of predicted antigenic peptides.
Sl. No. Peptide No. of
residues
1 NH2-TILRLAITSAVHKARQSYGD-COOH 20
2 NH2-VRQLQKDLASNIIVSKHSVD- COOH 20
3 NH2-RVGNAAFASYFAKYVPNTIR- COOH 20
Purification and heterologous expression of RBL
73
Amino acid
0 20 40 60 80 100 120 140 160 160 180 200 220 240 260 260 280 300
0
-1
-2
-3
3
2
1
An
tig
en
ic i
nd
ex
30.0 35.0 40.0 45.0 50.0 55.0 60.0 min
0
25
50
75
100
125
150
175
200
225
250
275
mAU
Ch2-280nm,4nm (1.00) Ch1-230nm,4nm (1.00)
45.406
46.962
48.628
34.444
40.111
41.505
44.188
45.552
46.965
54.534
Min
mA
U
Synthesis and purification of the peptides
The peptide NH2-CTILRLAITSAVHKARQSYGD-COOH with the highest
antigenecity score in Oryza sativa cv Japonica, TAG lipase sequence (T147
- D166
of
GenBank Acc. No. BAC83592.1) was assembled by Fmoc solid phase peptide
synthesis (Section 2.2.10). A cysteine residue was added to the NH2-terminus of the
peptide to promote the tagging of the peptides to the adjuvants. The peptide was
purified by RP-HPLC on a semi-preparative Shimpak C18 column (Figure 3. 10).
Figure 3. 9. Antigenicity plot of RBL as predicted by the algorithm of Hopp and Woods
(http://www.bioinformatics.org/JaMBW/3/1/7/).
Figure 3. 10. RP-HPLC profile of synthesized peptide resolved on a C18 Shimpak column
[250 mm × 21.2 mm (i.d.), 10 µ] using a binary gradient of 70 % acetonitrile containing
0.05 % TFA and water containing 0.1 % TFA.
Purification and heterologous expression of RBL
74
2304
m/z500 520 540 560 580 600 620 640 660 680 700 720 740
%
0
100
19071103 95 (1.809) TOF MS ES+ 6.36e3577.00
576.75
576.51
577.26
577.51
577.75
0 10 20 30 40 50 60 70 80
min
0
25
50
75
100
125
150
175
200
225
250
275
300
325
mA
U
46.1
63
The peptide that eluted at 45.552 minutes was collected over several runs. The
purified peptide was analyzed by analytical RP-HPLC. The homogeneity of the
purified peptide is shown in the Figure 3. 11. The results show that the purity of the
peptide was >99 %. The mass of the synthesized peptide was determined by ESI-MS
and a mass of 2,304 Da was obtained (Figure 3. 12). Further, the sequence of the
synthesized peptide was validated by amino acid analysis and NH2-terminal
sequencing. The RP-HPLC profiles of the PTH-amino acids released in the first four
cycles of Edman degradation correspond to CTIL, validating the sequence of the
synthesized peptide (Figure 3. 13).
Figure 3. 11. RP-HPLC profile of the purified peptide. The peptide purified by semi-
preparative RP-HPLC was analyzed using a Discovery C18 column (4.6 250 mm, 5 µm) on a
Waters Associate HPLC, using a linear gradient of 0.1 % TFA and 70 % acetonitrile
containing 0.05 % TFA.
Figure 3. 12. ESI-MS spectrum of synthetic peptide showing m/z of 577 and mass 2304
Da.
Purification and heterologous expression of RBL
75
T
I
L
39.6
30.4
21.2
12.0
2.8
mV
5.0 10.0 15.0 20Minutes
39.6
30.4
21.2
12.0
2.8
mV
5.0 10.0 15.0 20Minutes
39.6
30.4
21.2
12.0
2.8
mV
5.0 10.0 15.0 20Minutes
39.6
30.4
21.2
12.0
2.8
mV
5.0 10.0 15.0 20Minutes
Figure 3. 13. RP-HPLC elution profile of the PTH-amino acids released from purified
peptide by the first four cycles of Edman degradation by automated gas phase
sequencing (Section 2.2.12). The eluted peaks were compared with the standard profile to
deduce the sequence.
Purification and heterologous expression of RBL
76
A B
BA C
1
2
3
Lipase-I
RBL (Lipase-II)
1 2 3
Immuno-detection of RBL
The anti-RBL polyclonal antibodies raised against the synthetic peptide in
New Zealand White rabbits were used to detect lipase. The crude extract of rice bran
and the purified RBL following native-PAGE were electro blotted on a nitrocellulose
membrane and probed with the polyclonal antibodies raised against the synthetic
peptide. Native-PAGE of the crude extracts of rice and rice bran followed by enzyme
staining revealed two different lipase species corresponding to Lipase-I and RBL
(Lipase-II) (Figure 3. 14A). The migration of two lipase species of rice extracts are
consistent with that of the two lipases reported in rice bran (Figure 3. 14A). The anti-
RBL antibodies showed cross reactivity to both Lipase-I and RBL (Figure 3. 14B).
The antibodies were very specific to the peptide and did not cross react with bovine
serum albumin (Figure 3. 14C). The cross reactivity of the anti-RBL with Lipase-I
and RBL reveal that these two lipases have similar antigenic determinants. A single
cross reactive species was observed in the purified RBL also indicating the
homogeneity (Figure 3. 15).
Figure 3. 14. Native-PAGE profile of purified RBL stained for A) Lipase activity. Lane
1, crude extract of rice; lane 2, rice bran and lane 3, purified RBL. B) Crude extracts of rice
probed with anti-RBL and C) Dot blot analysis: 1) BSA, 2) peptide and 3) rice crude
extract.
Figure 3. 15. Native-PAGE profile of the purified rice bran lipase stained for A) lipase
activity and B) immuno-detection with anti-RBL antibodies.
Purification and heterologous expression of RBL
77
28S
18S
1 2
To attribute the physiological properties of lipase to its function requires
information on the three dimensional structure. To better understand and correlate the
structure-function relationship at the molecular level, the gene encoding the RBL was
cloned in a bacterial expression system.
Isolation of total RNA
Total RNA was isolated from Oryza sativa cv Indica var. IR64 seeds using
TRIzol reagent following the manufacturer’s instructions for plant tissue. The quality
of the isolated RNA was analyzed on denaturing agarose gel electrophoresis (Figure
3. 16). The presence of two distinct bands of 28S and 18S RNA indicated that the
RNA preparation was suitable for further studies. The A260/280 ratio of the purified
RNA was 2.0-2.1 indicating the purity and absence of DNA.
cDNA synthesis
First-strand cDNA was synthesized by reverse transcribing 10 µg of total
RNA using a High Capacity cDNA Archive kit. The amplifiability of the prepared
cDNA was analyzed by amplification of the actin gene. The reaction set up with total
RNA, without the addition of reverse transcriptase served as the control.
Contaminating DNA was evaluated by PCR amplification using control RNA as
template and actin gene specific primers (Table 2. 5). The absence of amplification
products indicated the absence of contaminating DNA (Figure 3. 17, Lanes 1-2). The
amplification of a 580 bp fragment of the housekeeping gene, actin showed that the
cDNA was intact (Figure 3. 17, Lanes 5-7) and suitable for use in further
amplifications. The synthesized cDNA were stored at -20 C.
Figure 3. 16. Denaturing agarose gel electrophoresis profile of total RNA isolated from
the rice (Oryza sativa) seeds. Lanes 1-2, total RNA.
Purification and heterologous expression of RBL
78
1 2 3 4 5 6 7
580 bp
987 bp
M 1 2 3 Pc
Figure 3. 17. Agarose gel electrophoresis profile showing amplification of actin, the
house keeping gene. Lane 1, Control (without reverse transcriptase); lane 2, negative control
(without RNA); lane 3, premise control; lane 4, 100 bp DNA molecular marker and lanes 5-7,
580 bp PCR amplification product of actin.
Figure 3. 18. Amplification of RBL gene using the synthesized cDNA. Lane M, 100 bp
DNA molecular marker; lanes 1-3, RBL and lane Pc, premise control.
Purification and heterologous expression of RBL
79
Amplification of RBL gene
The primers for the amplification of open reading frame of RBL was designed
based on the determined NH2-terminal sequence (ASHGRDIS…) and sequence of the
retrieved putative TAG lipase (GenBank Acc. No. BAC83592.1, Table 2. 5). Using
the primer pair RBL 987F/R, a 987 bp amplicon was amplified. The thermal cycling
conditions included an initial denaturation at 98C for 30s followed by 30 cycles of
98C for 10s, 60C for 30s, 72C for 30s and then a final extension at 72C for 7
min. The PCR product was evaluated by 2 % agarose gel electrophoresis and
visualized (Figure 3. 18). The amplified product was purified and subjected to direct
DNA sequencing using a set of primers as described in Section 2.2.20. A BLAST
search of the obtained sequence was homologous with the putative TAG lipase
(GenBank Acc. No. BAC83592.1) (Figure 3. 19). A pair wise sequence alignment of
the RBL gene with the putative TAG lipase showed that these two sequences were
identical.
Construction of expression vector pRSET ALip
The E. coli expression vector pRSET A was used to construct pRSET ALip by
directional cloning using BamH1 and EcoR1 restriction sites. The multiple cloning
site of pRSET A vector is as illustrated in Figure 3. 20. The restriction sites for
BamHI and EcoRI were engineered at 5' and 3' ends of the 987 bp amplicon using the
primer pair LipClF/R and 987 bp product as template. The thermal cycling conditions
included an initial denaturation at 98C for 30s followed by 25 cycles of 98C for
10s, 62C for 30s and 72C for 30s followed by a final extension at 72C for 7 min.
The amplified 1007 bp product was purified by 2 % agarose gel electrophoresis
(Figure 3. 21). The purified amplicon and pRSET A DNA were simultaneously
digested with BamHI and EcoRI following the manufacturer’s instructions. The
digested products were purified and ligated using T4 DNA ligase. The ligated
products were transformed into chemically competent E. coli strain DH5 to generate
a recombinant vector named pRSET ALip (Figure 3. 22).
Pur
ific
atio
n an
d he
tero
logo
us e
xpre
ssio
n of
RB
L
80
Fig
ure
3.
19. M
ult
iple
seq
uen
ce a
lig
nm
ent
of
seq
uen
ce o
bta
ined
fro
m R
BL
wit
h p
uta
tive
TA
G l
ipa
se (
Gen
Ba
nk
Acc
. N
o. B
AC
83
59
2.1
).
Purification and heterologous expression of RBL
81
Bam H1 EcoR 1
RBL
1 2 3 4
1007 bp
Figure 3. 20. Vector map and multiple cloning site of vector pRSET A.
Figure 3. 21. Agarose gel electrophoresis of PCR products with restriction sites
engineered. Lane 1, 100 bp DNA molecular marker and lanes 2-4, LipCl.
Figure 3. 22. Schematic representation of the strategy used for directional cloning of
RBL gene in pRSET A.
Purification and heterologous expression of RBL
82
1 2 3 4
L 1 2
1000 bp
500 bp
7 L 8 9
1169 bp
A B C
3 4 5 6
The colonies harboring the plasmid were isolated on LB-agar plates
supplemented with 100 µg/mL ampicillin. Plasmid DNA was isolated from individual
colonies and subjected to 0.8 % agarose gel electrophoresis. The recombinant clones
harboring RBL were assessed by gel shift assay (Figure 3. 23). The insertion of gene
of interest was further confirmed by insert release upon digestion with BamH1 and
EcoR1 (Figure 3. 24A) and linearization using the restriction enzyme EcoRV (Figure
3. 24B). A PCR amplification using vector and gene specific primers
(T7upF/RBL987R) showed a product of expected size 1169 bp (Figure 3. 24C). All
these results indicate the identified clones contain the inserted 987 bp RBL gene. The
plasmid was named pRSET ALip.
Figure 3. 23. Agarose gel electrophoresis profile of plasmid DNA isolated from
transformed E. coli strain DH5. Lane 1, pRSET A and lanes 2-4, E. coli DH5
transformed with pRSET ALip. A gel shift is observed in lanes 2 and 3.
Figure 3. 24. Agarose gel electrophoresis profile of pRSET ALip: A) digested with
BamH1 and EcoR1. Lane L, 100 bp DNA molecular marker and lanes 1-2, pRSET ALip. B)
EcoRV. Lane 3, undigested pRSET ALip; lane 4, pRSET A and lanes 5-6, digested pRSET
ALip and C) PCR amplified products using the primer pair T7 upF and RBL 987R. Lane
7, premise control and lanes 8-9, pRSET ALip.
Purification and heterologous expression of RBL
83
gcttcccatgggagagatatctctgtccagcactctcagcaaactttgaactatagccat
A S H G R D I S V Q H S Q Q T L N Y S H
actcttgccatgactcttgtggaatatgcttctgctgtgtacatgacagatttaacagct
T L A M T L V E Y A S A V Y M T D L T A
ctttatacatggacgtgctcaaggtgtaatgacttgactcaaggctttgagatgaaatct
L Y T W T C S R C N D L T Q G F E M K S
ctaatcgtggatgtggagaactgcctacaggcattcgttggtgtggattataatttaaat
L I V D V E N C L Q A F V G V D Y N L N
tcaataattgttgcaataagaggaactcaagaaaacagtatgcagaattggatcaaggac
S I I V A I R G T Q E N S M Q N W I K D
ttgatatggaaacaacttgatctgagctatcctaacatgcctaacgcaaaggtgcacagt
L I W K Q L D L S Y P N M P N A K V H S
ggatttttctcctcctataataacacgattttacgtctagctatcacaagtgctgtccac
G F F S S Y N N T I L R L A I T S A V H
aaggcaagacagtcatatggagatatcaatgtcatagttacagggcactcaatgggagga
K A R Q S Y G D I N V I V T G H S M G G
gccatggcatccttctgtgcgcttgatctcgctatcaatcttggaagcaatagtgttcaa
A M A S F C A L D L A I N L G S N S V Q
ctcatgactttcggacagcctcgtgttggcaatgctgcttttgcctcttattttgccaaa
L M T F G Q P R V G N A A F A S Y F A K
tatgtgcccaacacgattcgagtcacacatggacatgatattgtgccacatttgccccct
Y V P N T I R V T H G H D I V P H L P P
tatttctcctttcttccccatctaacttaccaccacttcccaagagaggtatgggtcaat
Y F S F L P H L T Y H H F P R E V W V N
gattctgagggcgacataaccgaacagatatgtgatgatagtggtgaagatccaaattgc
D S E G D I T E Q I C D D S G E D P N C
tgcaggtgcatctccacatggagtttgagcgttcaagaccatttcacatacctgggagtt
C R C I S T W S L S V Q D H F T Y L G V
gatatggaagctgacgactggagcacttgtagaatcatcacagctgaaaatgttaggcaa
D M E A D D W S T C R I I T A E N V R Q
ctccaaaaggatctcgccagcaacatcatcgtctccaagcactctgtcgatgtcactatt
L Q K D L A S N I I V S K H S V D V T I
gtagaacctagttcacaaacatattga
V E P S S Q T Y -
Figure 3. 25. The complete cDNA sequence of the open reading frame of RBL and the
translated amino acid sequence. The underlined sequences denote the sequences of tryptic
peptides obtained by Edman degradation.
The sequence of the inserted 987 bp fragment was analyzed for any mutation
and confirmed by Big-dye terminator di-deoxy sequencing on an automated DNA
sequencer (ABI 310 DNA Genetic Analyzer, Applied Biosystems, Foster City, CA,
USA). The open reading frame of 987 bp obtained was translated into the
corresponding protein sequence. The nucleotide sequence and translated sequence are
shown (Figure 3. 25).
Purification and heterologous expression of RBL
84
63
14.3
20.1
29
43
kDa
97.4
1 2 3 M 4 5
1 2 3 4 M 1 2 3 4
6.5
14.3
20.1
29
43
Insoluble Soluble
kDa
Figure 3. 26. SDS-PAGE (12.5 % T, 2.7 % C) profile of cell free extracts of various E.
coli strains transformed with pRSET ALip. Lane 1, undinduced; lane 2, BL21(DE3)pLysS;
lane 3, Origami(DE3)pLysS; lane 4, RIL(DE3)pLysS; lane 5, Rosetta(DE3)pLysS and lane
M, molecular weight markers: phosphorylase b (97,400 Da), BSA (66,000 Da), ovalbumin
(43,000 Da), carbonic anhydrase (29,000 Da) and soybean trypsin inhibitor (20,000 Da).
Arrow denotes the expressed protein.
Figure 3. 27. SDS-PAGE (12.5 % T, 2.7 % C) profile of cell free extracts of rRBL
expressed in RIL(DE3)pLysS. Lane 1, pRSETA; lane 2, uninduced; lane 3, induced with
0.3 mM IPTG; lane 4, induced with 0.5 mM IPTG and lane M, low range protein molecular
marker: ovalbumin (43,000 Da), carbonic anhydrase (29,000 Da), soybean trypsin inhibitor
(20,000 Da), lysozyme (14,300 Da) and aprotinin (6,500 Da).
Purification and heterologous expression of RBL
85
Expression of RBL in E. coli
Protein expression in four different E. coli systems was investigated. The
expression vector pRSET ALip was transformed into BL21(DE3)pLysS,
Origami(DE3)pLysS, RIL(DE3)pLysS, and Rosetta(DE3)pLys. Prolonged incubation
after IPTG induction was performed at lower temperatures to partly compensate for a
slower growth rate. The cells were harvested at 5,000 g for 10 min at 4 C and lysed
in 50 mM sodium phosphate buffer pH 7.4 by sonication. The supernatant and pellet
were analyzed for the recombinant protein by lipase activity and SDS-PAGE (12.5 %
T, 2.7 % C). BL21(DE3)pLysS transformed with pRSET A was used as the control
(Figure 3. 26, Lane 1). Insignificant expression in BL21(DE3)pLysS and
Origami(DE3)pLysS was observed (Figure 3. 26, Lane 2). Prokaryotes often differ in
their codon preferences from eukaryotes when expressing heterologous proteins.
Therefore two other strains Rosetta(DE3)pLysS and RIL(DE3)pLysS were used for
heterologous expression. These strains harbor a plasmid encoding cognate tRNAs for
rare codons. A significantly higher level of rRBL was expressed in RIL(DE3)pLysS
(Figure 3. 26, Lane 4) and Rosetta(DE3)pLysS (Figure 3. 26, Lane 5) at 16 °C in
comparison to 37 °C. Despite the compensation in cultivation time, the over-
expressed protein was obtained as insoluble inclusion bodies enriched in the cell
lysate pellet (Figure 3. 27, Lanes 2-4). Leaky expression was observed in the
uninduced culture. No lipase activity was measurable in the expressed protein,
although it accounted for >50 % of the total protein.
In-gel trypsin digestion
In order to confirm whether the expressed protein was RBL or not, in-gel
digestion of the expressed protein and analysis of peptide sequences was attempted.
The inclusion bodies were separated by SDS-PAGE (12.5 % T, 2.7 % C) and the
expressed protein digested with TPCK-trypsin. The resulting tryptic peptides were
resolved and purified by RP-HPLC (Figure 3. 28). The sequences of the major
peptides were obtained by Edman degradation. The sequences of two peptide
fragments (peak 2: YVPN and peak 10: VTHGHDIVPHLPPYFSFLPH) obtained by
Edman analysis correspond to residues 201 to 205 and 208 to 227 of the translated
protein sequence (Figure 3. 25). These results show that the protein expressed was
RBL and not otherwise. The absence of lipase activity in the expressed protein is
probably due to incorrect folding.
Purification and heterologous expression of RBL
86
Minutes
0 5 10 15 20 25 30 35 40 45 50 55 60
mA
U
0
25
50
75
100
mA
U
0
50
75
100
1
2
34
56
7
9
10
11
12
13
8
Peak 2 : YVPNT
Peak 10 : VTHGHDIVPHLPPYFSFLPH
Figure 3. 28. RP-HPLC profile of tryptic peptides of rRBL on a Discovery C18 column
(4.6 250 mm, 5 µm) detected at 230 nm. Inset shows the sequences of peptides (peak 2
and 10) obtained by Edman degradation.
Refolding of rRBL
Refolding of the rRBL in solution was carried out to render the protein
functional. The insoluble fraction was resuspended in 100 mL of lysis buffer (50 mM
sodium phosphate buffer, pH 7.4 containing 0.1 M NaCl and 0.5 % Triton X-100) and
disrupted by intermittent sonication on ice. The inclusion bodies were recovered by
centrifugation at 10,000 g for 20 min at 4 °C. After another wash with 100 mL lysis
buffer and intermittent sonication, the inclusion bodies were solubilized in 20 mL of
the denaturing buffer (50 mM sodium phosphate buffer containing 6 M guanidinium
hydrochloride and 50 mM DTT, pH 7.4). The final protein concentration was 10
mg/mL. Refolding was performed by dilution with 1 L of the activation buffer (50
mM sodium phosphate buffer, pH 7.4 containing 2 M urea, 0.5 M oxidized
glutathione and 1 mM reduced glutathione) and incubation at 4 °C for 15 h. The
solution was concentrated by ultra filtration with a 10,000 Da cut-off cellulose acetate
flat membrane on a Mini LabscaleTM
TFF system (Millipore Systems, Bedford, MA,
USA). The insoluble aggregates were removed by centrifugation at 20,000 g at 4
°C. The supernatant was used for analysis. The lipase activity of the refolded rRBL
was 0.013 U/mg as compared to 0.003 U/mg measured in the crude. These results
show that refolding had little effect on rRBL activity.
rRBL present in the inclusion bodies was purified by Ni-Sepharose affinity
chromatography. The rRBL-(His)6 tagged fusion protein eluted as a symmetrical peak
(Figure 3. 29). However none of the peak fractions showed any hydrolytic activity
Purification and heterologous expression of RBL
87
0 1 2 3 4 5
1.2
1.4
1.6
1.8
2.0
y = -0.193x + 2.091
R² = 0.994
Lo
g M
r
Migration rate (cm)
rRBL
C
20.1
29
43
kDa
M 1
A14.3 B
0 10 20 30 40
0.2
0.4
0.6
0.8
1.0
A2
80
nm
Elution volume (mL)
elu
tio
n
with either pNPA or triacetin. SDS-PAGE analysis revealed a single protein species
(Figure 3. 30A). The molecular mass of rRBL was 40,000 ± 1,500 Da. The
appearance of a protein of the molecular size of RBL (with added His tag) that cross
reacted with RBL antibodies demonstrate that the purified protein was rRBL (Figure
3. 30B).
Figure 3. 29. Immobilized metal ion chromatography elution profile of rRBL. The arrow
indicates the start of elution with 0.5 M imidazole.
Figure 3. 30. SDS-PAGE (12.5 % T, 2.7 % C) profile of purified rRBL expressed in
RIL(DE3)pLysS. A) Stained for protein CBB R-250; B) immuno-detection of rRBL with
anti-RBL antibodies and C) Plot of Log Mr of the marker proteins vs migration rate in
SDS-PAGE to determine molecular mass.
Purification and heterologous expression of RBL
88
1 2 3 4 1 2 3 4 5
A B
1 L 2 3 4
C
987 bp
63
14.3
20.1
29
43
kDa
97.4
M 1 2
Figure 3. 31. Agarose gel electrophoresis profile of plasmid DNA isolated from
transformed E. coli DH5. A) pGEX4T-2Lip and B) pET20b(+)Lip. Lane 1, control
vector and lane 2-5, transformed DH5. C) PCR amplification of RBL gene using putative
clone as template. Lane 1, premise control; lane L, 100 bp DNA molecular marker; lane 2,
pGEX4T-2Lip; lane 3, pET20b(+)Lip and lane 4, positive control.
Figure 3. 32. SDS-PAGE (12.5 % T, 2.7 % C) profile of cell free extracts of
RIL(DE3)pLysS. Lane 1, transformed with pGEX4T-2Lip; lane 2, transformed with
pET20b(+)Lip and lane M, molecular weight markers.
To assess the effect of Glutathione-S-transferase (GST) fusion tag on
solubility and/or functionality of the expressed RBL, the gene was sub cloned into
SmaI site of pGEX4T-2 vector. The RBL gene was also cloned into EcoRV site of
pET20(b)+ vector that direct the expressed protein into periplasmic space of E. coli,
which is known to enhance the solubility of the expressed protein. The ligated
products were transformed into chemically competent E. coli DH5 cells. The
plasmid DNA was isolated from individual colonies and subjected to agarose gel
electrophoresis. The putative clone was screened by gel shift assay and confirmed by
PCR amplification using the RBL gene specific primers (Figure 3. 31). Protein
expression either with GST fusion tag or directing the expressed protein to
periplasmic space in RIL(DE3)pLysS was found to be insignificant (Figure 3. 32).
Purification and heterologous expression of RBL
89
Discussion
Several lipases of both plant and animal origin have been purified and their
biochemical and kinetic properties elucidated in detail. The lipases of plant origin
include corn scutella (Lin & Huang, 1984), castor bean (Maeshima & Beevers, 1985),
Vernonia galamensis (Ncube et al., 1995) and rice bran (Funatsu. et al., 1971; Aizono
et al., 1976; Rajeshwara & Prakash, 1995; Bhardwaj et al., 2001). Although several
plant lipases have been characterized, information on cloning and heterologous
expression are limited (Aloulou et al., 2006; Yu et al., 2007; Larsen et al., 2008; Sabri
et al., 2009 ). Rice bran is an abundant by product during milling and a rich source of
proteins, vitamins and fat. The lipases of rice bran cause rapid deterioration of both oil
and bran (Prakash, 1996). Initial biochemical investigations of rice bran lipases were
carried out in the early 1970s by Funatsu et al., (1971) and Aizono et al., (1976). Over
the past two decades, rice bran lipases have drawn the attention of a wide range of
disciplines due to their importance in industrial applications. Protein biochemists use
lipase as a novel protein to explore lipid biotranformation reactions due to its potential
substrate selectivity, 1, 3-regioselectivity and enantioselective properties (Mukherjee,
1994). More detailed knowledge of RBL at the molecular level would provide
valuable information that would aid in elucidating the physiological functions
attributed to it. Although several lipases have been identified in rice bran, none of
them have been cloned and over expressed and their functionality demonstrated. A
lipase cloned from a rice seed coat cDNA library encoding 361 amino acid residues
had a molecular mass of 40,000 Da (Kim, 2004). A novel 27,000 Da recombinant
esterase of rice bran OsEST-b was shown to be distinct from traditional esterases and
lipases (Chuang et al., 2011). We report here the purification of RBL with higher
specific activity, in order to make available a homogenous preparation for detailed
biochemical characterization. Molecular cloning of the RBL gene and its expression
in heterologous host E. coli is reported.
Purification methods used have generally depended on nonspecific techniques
such as precipitation, hydrophobic interaction chromatography, gel filtration, and ion
exchange chromatography. Affinity chromatography has been used in some cases to
reduce the number of individual purification steps needed. In this study, a
combination of procedures, ion exchange chromatography and size exclusion
chromatography have been used to purify RBL to homogeneity. RBL interacts with
DEAE Sepharose at pH 7.4 and was used effectively to purify it. The bound RBL was
Purification and heterologous expression of RBL
90
selectively eluted with 0.2 M KCl. The size exclusion chromatography step on
Sephadex G-75 (Figure 3. 2) restricted the contaminating proteins and helped increase
the specific activity to 189.7 ± 7.9 U/mg (Table 3. 1). A single lipase species was
observed in Native-PAGE detected by activity staining (Figure 3. 3) as well as SDS-
PAGE analysis (Figure 3. 4). Molecular weights of plant lipases are diverse and
variable. The purified lipase had a molecular mass of 35,293 Da as revealed by ESI-
MS (Figure 3. 5). The homogeneity was also revealed by the release of a single amino
acid Ala during NH2-terminal sequencing using Edman degradation and a single
homogenous peak by RP-HPLC (Figure 3. 6). The pI of the RBL was found to be
9.14 (Figure 3. 7). The molecular weight and pI are similar to the reported lipase
(Aizono et al., 1976). Polyclonal antibodies raised against a synthetic peptide cross
reacted with the purified RBL (Figure 3. 15). All these results assured that the
preparation was homogenous and could be used for functional studies and
biochemical characterizations. Lipases show varying degrees of lipid class selectivity,
regioselectivity, fatty acid selectivity and stereoselectivity (Ncube et al., 1995). Our
observations indicate that the selectivity of RBL is towards rice bran oil. The results
on chain length selectivity, biochemical characteristics and kinetics of RBL are
presented and discussed in Chapter IV.
Isolation of the genes encoding lipases has provided information on their
number and organization as well as probes for studying the regulation of their
expression. The amplified 987 bp fragment produced by PCR reaction using a rice
seed cDNA as template covered the ORF of the entire RBL gene devoid of its signal
sequence. The DNA sequence analysis of the plasmid pRSET ALip indicated a
coding region for 328 amino acids. The translated amino acid sequence of rRBL was
similar to the amino acid sequence of putative TAG lipase (GenBank Acc. No.
BAC83592.1) (Figure 3. 25).
Expression of rRBL was almost absent in the host E. coli BL21(DE3)pLysS
(Figure 3. 26). Prokaryote and eukaryotes differ in their codon usage, which is a
major bottleneck during translation. This possibly explains the near absence of rRBL
expression in E. coli. Examination of the 987 bp sequence revealed that the codons
used for Arg (AGA, AGC), Pro (CCC) and Gly (GGA) occur 19 times out a total of
328 codons. These codons are rarely used by E. coli. The strategies to minimize such
transcriptional precints include either codon optimization (Ferrer et al., 2009) or
changing the host strains, temperature variation or plasmid compensation for rare
Purification and heterologous expression of RBL
91
tRNA (Sorensen & Mortensen, 2005). Investigating the effect of different host strain
seemed obvious. No significant difference in the expression level was observed using
the strain Origami(DE3)pLysS (Figure 3.26). The E. coli strains supporting the
formation of disulphide bonds (Origami(DE3)) was also not suitable for the
expression in strains supplementing rare codons. Induction in the E. coli strain
RIL(DE3)pLysS and Rosetta(DE3)pLysS showed overexpression with rRBL
accounting for >50 % of total protein (Figure 3. 26). Therefore supplying rare codons
increased the expression level. Temperature had a profound effect on the level of
rRBL expressed. The fact that lowering the temperature to 20 C had an impact on the
achieved rRBL expression supports the hypothesis that the translation is a hurdle in
the expression of rRBL. The expressed protein rRBL was found in the insoluble
inclusion bodies. A rice bran esterase expressed in E. coli Tuner(DE3)pLysS was
mainly recovered in the soluble fraction (Chuang et al., 2011). The expressed rRBL
purified from the cell lysate neither hydrolyzed p-nitrophenyl esters nor triacetin. The
very high level expression of RBL (Figure 3. 28) could yet be another probable cause
for aggregation and accumulation in inclusion bodies, leading to an inactive lipase.
No substantial increase in lipolytic activity of rRBL was evident upon unfolding and
refolding. No beneficial value of refolding rRBL was found nor did cultivation at
reduced temperature limit the in vivo aggregation of rRBL. The reports on the cloning
and expression of various lipases also demonstrate that in almost all the strategies
used, the proteins were expressed as fusion proteins and required either an activation
process, or co-expression of chaperone proteins or co-lipase for the functional
expression (Cui et al., 2011). Our efforts on the expression of RBL with GST as the
NH2-terminus fusion partner or targeting the expressed protein to periplasmic space to
enhance solubility of aggregation prone rRBL failed to obtain a functional enzyme.
Pichia pastoris, a methylotropic yeast is often used to overcome codon bias and
hurdles of post translational modifications in prokaryotes and is considered as an
excellent host for production of plant proteins. The molecular cloning and functional
expression and characterization of rRBL as a secretory protein in P. pastoris are
discussed in detail in the following chapter IV.