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Expression of an aphid-induced barley methyltransferase in Escherichia coli , purification and characterisation of the enzyme. by Irene Ingvor Zetterlund. Aim. To test the hypothesis that OMT is involved in gramine biosynthesis

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

by Irene Ingvor Zetterlund

slide2
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
background barley hordeum vulgare
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
background bird cherry oat aphid rhopalosiphum padi
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
background plant defence reactions
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
background plant defence reactions gramine
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

background omt
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
hypothesis
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
methods impact cn protein purification system
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
methods
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)
materials growth and treatment of plants
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
results synthesis of the coding region of the omt gene
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
results cloning of the omt gene into the ptyb 12 vector
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

results transformation of e coli dh5 t1 and screening for recombinants
Results: Transformation of E. coliDH5α-T1 and screening for recombinants
  • Toamplify the OMT sequence E. coliDH5α-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

results control of the inserts
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
results control of the insert
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

results transformation of e coli er2566 and screening for recombinants
Results: Transformation of E. coliER2566 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

results induction of protein expression
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

results optimizing of the protein induction conditions
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

results western blot
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.

results purification of the target protein
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

results purification of the target protein conditions
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 1Concentration of the target protein

results silica gel thin layer chromatography
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 – SAM+3H-SAM (95+5)
    • 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

results assay of the methyltransferase activity
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

results assay of the methyltransferase activity1
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

discussion transformation of e coli dh5 t1
Discussion: Transformation of E. coliDH5α-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
discussion assay of the methyltransferase activity
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
discussion assay of the methyltransferase activity1
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.
summary
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%
conclusion
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
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