Expression of an aphid-induced barley methyltransferase in Escherichia coli, purification and characterisation of the enzyme

1
Expression of an aphid-induced barley
methyltransferase in Escherichia coli,
purification and characterisation of the enzyme

• by Irene Ingvor Zetterlund

2
Aim

• To test the hypothesis that OMT is involved in
  gramine biosynthesis
• To clone the OMT gene into an expression vector
  with purpose to synthesize the enzyme in E. coli
• To characterize the enzyme kinetically

3
Background Barley (Hordeum vulgare)

• Is an important cereal in Sweden
• Is cultivated on the large area of arable land,
  about 400 000 ha
• Is used in the malting industry and for livestock
  feed
• All kind of farm animals can be fed on it

4
Background Bird cherry-oat aphid (Rhopalosiphum
padi)

• One of the most serious barley pests
• Transmit virus infections, i. a. barley yellow
  dwarf virus
• Phloem-feeding insects, cause little tissue
  damage
• Are perceived by plants as pathogens
• Breed by sexual reproduction and parthenogenesis
• Overwinter as eggs on its primary host, bird
  cherry
• In summer make use of diverse grasses as
  secondary hosts, among them barley

5
Background Plant defence reactions

• Alkaloids a big group of N-containing secondary
  metabolites, have strong physiological effects in
  defence against herbivores
• Aphids induce pathogen-defence response
• Jasmonic acid signalling pathway induces
  expression of a wide range of defense genes
• One of them was identified as an
  O-methyltransferase gene

6
Background Plant defence reactions - Gramine

• Gramine - indole proto-alkaloid, secondary
  metabolite in barley and some other species in
  the grass family Poaceae
• Induced in barley upon aphid infestation
• Found in epidermis and in mesophyll parenchyma
• Missing in the vascular bundles
• The higher gramine amount
• the lower vulnerability of
• barley to aphids
• Synthesized from tryptophan
• via 3-aminomethylindole
• NMT catalyzes SAM-dependent
• conversion of AMI to MAMI
• and from MAMI to gramine

7
Background OMT

• One gene, induced by the aphid, is encoding an
  O-methyltransferase, OMT
• It is also induced by the jasmonic acid
  signalling pathway
• OMTs generally
• methylate caffeic acid
• lead to lignin precursors
• or various classes of flavonoids
• But not all of barley cultivars had OMT gene in
  their genome
• In the barley varieties missing the gene, gramine
  was not found either
• In all gramine-containing lines OMT was present

8
Hypothesis

• The gene, characterized as encoding for an
  O-methyltransferase acting on caffeic acid,
• might actually be encoding an N-methyltransferase,
  involved in gramine biosynthesis

9
Methods IMPACT-CN Protein Purification System

• IMPACT intein mediated purification with an
  affinity chitin-binding tag
• A target protein is fused to a self-cleavable
  intein tag
• A chitin-binding domain in intein tag allows
  purification of the target protein on the chitin
  column
• The intein tag undergoes specific self-cleavage
  in presence of DTT
• The target protein releases from the chitin-bound
  intein tag

10
Methods

• RT-PCR and PCR
• Cloning of the target gene into the vector
• Transformation of the competent cells
• Agarose and SDS-PAGE gel electrophoresis
• Western blotting
• Bradford microassay for protein quantification
• Silica gel thin-layer chromatography (TLC)

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Materials Growth and treatment of plants

• H. vulgare, variety Lina, susceptible to the
  aphids
• Sown in November 2003
• Grown in a growth chamber at 26oC, long day, (18
  h light/6 h darkness)
• 5-day-old barley plants were harvested
• Their green tissue was treated with 45 µM
  jasmonic acid for 24 hours to induce the OMT-gene
  

12
Results Synthesis of the coding region of the
OMT gene

• The total RNA was isolated from barley green
  tissue
• RNA was reverse transcribed into single-stranded
  cDNA using the First-Strand Synthesis System for
  RT-PCR
• To amplify the coding region of the OMT gene by
  PCR primers OMTcloneF and OMTcloneR2 were used
• A product of about 1100 bp was visualized by 2
  agarose gel electrophoresis

13
Results Cloning of the OMT gene into the pTYB 12
vector

• The plasmid pTYB12, chosen as a vector - allows
  the fusion of the cleavable intein tag to the
  N-terminus of a target protein
• The plasmid - digested with
• the restriction nucleases SmaI
• and NdeI
• The DNA fragment - digested
• with restriction nuclease NdeI
• The digested DNA - ligated
• into the pTYB12 using the
• BioLabs Quick Ligation Kit

14
Results Transformation of E. coli DH5a-T1 and
screening for recombinants

• To amplify the OMT sequence E. coli DH5a-T1 were
  transformed with the new plasmid pTYB12-OMT
• The recombinant cells were selected on Petri
  dishes with LB medium containing 100 µg/ml
  ampicillin
• 96 randomly chosen colonies were
• inoculated in a microtitre plate in LB/amp
• medium
• PCR test for inserts using intern primers
• OMT F1 and OMT R1 obtained 3 clones

15
Results Control of the inserts

• To confirm the obtained recombinant clones,
  digesting reactions with restriction nucleases
  Kpn I, Nco I, Nde I and Sap I were carried out
  over night at 37oC
• The digested DNA was analyzed on 1 agarose gel
• Plasmid 1 gave the expected fragment pattern and
  thus was chosen as the pTYB12-OMT plasmid
• The digesting reaction with restriction
• nucleases resulted in bands as follow
• Kpn I - 6706 and 1801 bp
• Nco I - 7380, 680 and 447 bp
• Nde I - 8507 bp
• Sap I - 7810 and 697 bp

16
Results Control of the insert

• The plasmid was controlled for the right insert
  by PCR with 3 pairs of primers OMT clone F and
  OMT clone R2 (1) OMT F1 and OMT R1 (2), and
  Intein Forward and T7 Terminator Reverse (3)
• Bands of the correct sizes were visible on 2
  agarose gel, lane 1- 1100 bp, 2 - 348 bp and 3 -
  1300 bp
• To make sure that there was no error in the
  sequence of the cloned fragment, the plasmid
  pTYB12-OMT was sequenced at Cybergene
• The sequence proved to be identical to the
• one published earlier

17
Results Transformation of E. coli ER2566 and
screening for recombinants

• The E. coli strain ER2566 was provided by
  Impact-CN as a host strain for the expression of
  a target gene cloned in the pTYB12 vector
• ER2566 have a chromosomal copy of the T7 RNA
  polymerase gene inserted into the lacZ gene, and
  therefore under the control of the lacZ promoter
• Expression of T7 RNA polymerase is suppressed in
  the absence of IPTG, by the binding of lac I
  repressor to the lac promoter
• The transcription of the fusion protein takes
  place
• when IPTG is accessible
• Transformed cells ER2566 were selected on Petri
• dishes with LB/amp medium
• To control the protein induction ER2566 was
• transformed with the pMYB5 vector

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Results Induction of protein expression

• Induced with 0,5 mM IPTG at RT O/N
• SDS-PAGE analysis showed bands 100 kDa
• 100 kDa OMT-intein fusion protein
• Positive control - ER2566 transformed with pMYB5
  vector
• Negative control
• uninduced E1
• E2 and E6 chosen to
• continue the experiment

19
Results Optimizing of the protein induction
conditions

• Different conditions were verified
• Induction with 0,5 mM and 1 mM IPTG
• Temperature and time
• 37oC, 4 and 6 h
• RT, O/N
• 15oC, O/N
• SDS-PAGE analysis showed
• the strongest band about
• 100 kDa for the induction with
• 1 M IPTG at RT O/N

20
Results Western Blot

• Protein bands were transferred onto PVDF membrane
  by semi-dry transfer apparatus
• Immunoblotting
• primary antibodies - against the chitin binding
  domain
• Secondary antibodies - Goat Anti-Rabbit HRP
• The protein was detected using the ECL Plus
  Western Blotting kit and chemiluminescence in the
  CCD-camera
• The strongest bands of about 100 kDa
• in lanes 4 7
• E6 induced at RT with 1 mM IPTG
• showed the strongest band,
• conditions were the best for the protein
• expression.

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Results Purification of the target protein

• Purified using the IMPACT-CN Protein Purification
  System
• 1 l cell culture was induced with 1 mM IPTG at RT
  O/N
• Cells were broken by sonication
• Clarified cell extract, obtained by
  centrifugation, was loaded onto chitin column
• Cleavage reaction - started by
• adding Cleavage Buffer with DTT
• The protein was eluted using
• the Column Buffer
• SDS-PAGE analysis showed band
• 43 kDa corresponding to the purified
• methyltransferase

22
Results Purification of the target protein -
conditions

• Different conditions for the on column cleavage
  reaction were tested at 4oC and RT for 24 and 40
  hours
• Elution with the Column Buffer containing 0,5 M
  and 1 M NaCl
• The highest protein concentration elution with
  0,5 M NaCl
• The protein concentration was measured
  spectrophotometrically, using Bradford microassay
  method for protein quantification

Table 1 Concentration of the target protein
Cleavage reaction conditions Concentration of the target protein, µg/ml Concentration of the target protein, µg/ml Concentration of the target protein, µg/ml
Cleavage reaction conditions Eluant 1 Eluant 2 Eluant 3
RT, 24 h 240,00 18,80 0,00
RT, 40 h 162,12 31,14 4,81
4oC, 24 h 66,10 0,00 0,00
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Results Silica gel thin-layer chromatography

• For determination of the kinetic parameters of
  the methyltransferase were used as substrates
• AMI, MAMI and caffeic acid
• The methylation reactions were
• started and stopped by adding of
• Start SAM3H-SAM (955)
• Stop - stop buffer
• The methylation products were
• separated by means of TLC-
• plates standing in TLC solvent
• The regions with the reactions
• products were scraped from the
• TLC-plates for liquid
• scintillation counting

24
Results Assay of the methyltransferase activity

• Methyltransferase activity was measured by
  estimation of the amount of 3H-labelled product
  produced with methyl-3H-SAM
• 3H count per minute was calculated into built
  product per 1 mg protein

Table 2 AMI and MAMI methylation products built
per 1 mg protein, pmol/min, development in time
Table 3 AMI and MAMI methylation products built
per 1 mg protein, pmol/min, relative to the
substrates concentration
Incubation time, min 0 min 30 min 60 min
AMI 0 226,3 148
MAMI 0 52,6 71,2
Lina, AMI 0 - 16,6
Concentration, mM 0,75 mM 1,5 mM 3 mM
AMI 37,03 18,71 21,34
MAMI 2,38 30 1,53
25
Results Assay of the methyltransferase activity

• The reactions with the enzyme extract from barley
  green tissue did not show any activity
• The reactions with the methyltransferase purified
  by IMPACT-CN obtained some built product, but the
  data are questionable.

Table 5 Caffeic acid methylation products built
per 1 mg protein, pmol/min, development in time
pH 30 min 60 min
Enzyme 7,5 0 38,5
Enzyme 9,0 18,0 0
Lina 7,5 0 0
Lina 9,0 0 0
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Discussion Transformation of E. coli DH5a-T1

• E. coli had difficulties to survive after its
  uptake of the plasmid with the insert OMT
• Few recombinant colonies were obtained and the
  survivors turned out to have mutations in the OMT
  sequence
• The third transformation resulted in a frameshift
  mutation
• The fourth transformation was succesful

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Discussion Purification of the target protein
28
Discussion Assay of the methyltransferase
activity

• The methylation of AMI
• highest after 30 min incubation, decreasing later
  
• that contradicts the kinetic development in time
  as a logarithmic function
• The methylation of MAMI
• increases in time
• highest after 60 min incubation
• An explanation - the scraped samples were
  contaminated and thus are not trustworthy
• The methyltransferase activity was analyzed
  relatively to AMI and MAMI concentration
• production of MAMI from AMI is inversely
  proportional to the substrate concentration

29
Discussion Assay of the methyltransferase
activity

• Enzyme activity with caffeic acid as substrate -
  very little activity.
• The purified enzyme was going through several
  freeze-thaw cycles between the first measurement
  with AMI and MAMI as substrate and those with
  caffeic acid.
• This could have resulted in the loss of enzymatic
  activity.
• These experiments have to be repeated with
  freshly purified enzyme.

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Summary

• The enzyme exhibit little activity with caffeic
  acid but did methylate AMI and MAMI
• Thus it might be involved in gramine synthesis by
  methylating AMI and MAMI rather than acting as
  caffeic acid OMT
• Described as an O-methyltransferase, but a
  sequence similarity with other OMTs is only 40

31
Conclusion

• The enzyme carries out the transfer of a methyl
  group from S-adenosylmethionine to AMI,
  methylating it to MAMI and a methyl group from
  SAM to MAMI, with the formation of gamine, in
  fact acting as an N-methyltransferase in gramine
  biosynthesis
• This work supports the idea that the
  methyltransferase gene accession number U54767
  should be classified as an NMT-gene involved in
  gramine biosynthesis