RESULTS AND DISCUSSION 3. Purification of Rice Bran Lipase, …shodhganga.inflibnet.ac.in/bitstream/10603/36533/6/chapter 3.pdf · Cloning and Expression in E. coli . Purification

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.


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

(▪).


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.


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.


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.


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


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.


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.


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.


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.


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.


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.


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

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tive

TA

G l

ipa

se (

Gen

Ba

nk

Acc

. N

o. B

AC

83

59

2.1

).


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.


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.


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).


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).


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.


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


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.


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).


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


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


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.

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