Expression of an Aphid-Induced Barley Methyltransferase in Escherichia coli, Purification and Characterisation of the Enzyme

 

Ingvor Irene Zetterlund, Södertörn University College

Contents

 


Abstract 1

Introduction_ 2

Barley 2

Bird cherry-oat aphid_ 2

Plant defence reactions 3

Gramine 3

O-methyltransferase 4

Aim of the project 5

Materials and methods 5

Growth and treatment of plants 5

Total RNA isolation 5

RT-PCR_ 5

Cloning of the OMT gene into the pTYB12 vector 5

Transformation of E. coli DH5α-T1 and screening for recombinants  5

Transformation of the expression strain E. coli ER2566 and screening for recombinants  6

Induction of protein expression 6

Western blot 7

Purification of the target protein 7

Assay of the methyltransferase activity with AMI and MAMI 7

Assay of the methyltransferase activity with caffeic acid  8

Results 8

Cloning of the OMT gene into the pTYB 12 vector 8

Transformation of the expression strain E. coli ER2566  9

Induction of protein expression 10

Western blot 11

Purification of the target protein 11

Assay of the methyltransferase activity 12

Discussion_ 13

Cloning of the OMT gene into the pTYB12 vector 13

Transformation of E. coli and screening for recombinants  13

Transformation of the expression strain E. coli ER2566 and induction of protein expression  14

Purification of the target protein 14

Assay of the methyltransferase activity 14

Acknowledgements 15

References 15

 


Abstract

Barley (Hordeum vulgare) is the agricultural leading cereal; therefore it is of the high importance to keep the barley crop well protected from various pests and diseases. One of the most serios barley pests in Sweden, causing substantial economical losses due to expenses for insecticides, is the bird cherry-oat aphid (Rhopalosiphum padi). This aphid feeds on phloem sap, impairing plant develop­ment and transmitting virus infections, i. a. barley yellow dwarf virus. Inflicting little damage to plant tissue, aphids attack sets off the pathogen-defence response in barley. One gene, that has been shown to be induced by the aphid, is encoding an O-methyl­transferase, OMT (EC2.1.1.6, GeneBank accession U54767). This gene is also induced by the jasmonic acid signalling pathway, which medi­ates expression of defense genes. Previous studies demonstrated that not all barley cultivars had the OMT gene in their ge­nome, and in the va­rieties missing the gene, the barley indole alkaloid gramine was not found either. These facts have led to the hypothesis that the gene, which has been character­ized as coding for an O-methyltrans­ferase acting on caffeic acid, might actually be encoding an N-methyltransferase, in­volved in gramine bio­syn­thesis. Thus the charac­terization of this aphid-induced enzyme, with the pur­pose to find out its function in the undam­aged plant, has been the aim of the pro­ject.

 

Standard biotechnological methods were applied in this experiment, such as RT-PCR and PCR, Brad­ford microassay method for protein quantification, agarose and SDS-PAGE gel electrophoresis, Western blotting and TLC analysis. The coding sequence of the OMT gene was cloned into an expres­sion vector containing an intein tag. E.coli cells were transformed with the construct. The tar­get pro­tein was purified using a chitin column. SDS-PAGE analysis showed a major band at 43 kDa. Puri­fied protein fractions were used in enzyme assays with intermediates in the gramine biosynthesis pathway, AMI (3-aminomethylindole) and MAMI (N-methyl-3-aminomethylindole) as substrate, as well as with caffeic acid.  The enzyme activity with caffeic acid as substrate was obscure, the ex­periment results showed little activity, but they can be questioned. On the other hand both AMI and MAMI were acting as substrates and transformed to MAMI and gramine respectively. This work sup­ports the idea that the methyltransferase gene acces­sion number U54767 could be classified as an NMT-gene involved in gramine biosynthesis.

 

 


Introduction

The damage caused by herbivores to plants is de­pendent on the mode of herbivore feeding. Phloem-feeders produce little wound, in con­trast to chewing and cell-con­tent feeding in­sects, which cause more extensive tissue injury. Plant re­sponse is adjusted to different types of herbi­vore assault[1]. The pathogen-de­fence re­sponse pathways are induced by phloem-feed­ing in­sects, whereas chewing in­sects start out wound-signalling pathways. Among the nu­merous interac­tions between plants and insects, the interaction, cho­sen in these studies, is that between barley (Hordeum vulgare) and the bird cherry-oat aphid (Rhopalosiphum padi).

Barley

Barley (Hordeum vulgare) is an important ce­real in Sweden, cultivated on a large area of ar­able land, about 400 000 ha[2]. Barley is used in the malting industry as well as for live­stock feed. The barley cultivar used in this work, Lina, is a two-rowed spring-sown culti­var, which mainly is used for cattle feed. Bar­ley is the most common forage cereal and all kind of farm animals can be fed on it. There­fore it is of high significance to keep the barley crop well protected from various pests and dis­eases.

Bird cherry-oat aphid

One of the most serious barley pests, causing substantial economical losses due to insecti­cide expenses, is the bird cherry-oat aphid Rhopalosiphum padi. These aphids overwinter as eggs on its primary host, bird cherry (Prunus padus L.), and in summer make use of diverse grasses as secon­dary hosts, among them barley. Aphids are phloem-feeding insects. In contrast to the effect of chewing herbivores, tissue damage caused by aphids is not significant. Piercing plant foli­age with their stylet, aphids pass through the cuticle, epidermis and meso­phyll to attain veins of the phloem. Their stylet is in continuous contact with plant cells during feeding, which can con­tinue for several days1. Aphids do not only feed on the barley phloem sap and thereby withdraw nutrients from the plant; they can also transmit dif­ferent infec­tions. In special cells, mycetocytes, aphids carry bacteria, be­longing to the genus of Buchnera, which are synthesizing B vitamins for their host. These bacteria are transmitted maternally from parent to offspring and co-lonize the mycetocytes in the aphid embryo [3]. Buchnera have been im­plicated in promo­tion of aphid transmission of circulative viruses[4]. The most common viral disease of cereal crops, the barley yellow dwarf virus (BYDV), is transmitted by aphids. The BYDV symp­toms vary with the stage of crop development. In­fections at the seedling stage may result in death or dwarfing as well as ster­ile heads[5].

 

Aphids have a remarkable life cycle with re­gard to their ability of breeding by sexual re­production and parthenogenesis. In Sweden, the life cycle of R. padi is holocyclic, involving both parthenogenetic and sexual reproduction. The aphids overwinter as eggs on the winter host, bird cherry. In spring the eggs hatch, giving rise to several genera­tions of asexually reproducing females, vir­ginoparae[6]. A winged generation of females leaves bird cherry and moves to grasses, mainly spring-sown cereals. During summer they propagate parthenogenetically moving from one secon­dary grass host to another ac­cording to re­quirements. Population growth in aphids can be explosive. Under favourable en­vironmental conditions, a newly born aphid be­comes a re­producing adult within seven days, and can produce up to five offspring per day for up to 30 days. Aphids can develop wings for migra­tions, when their population increases and they have to compete for nutrients. In au­tumn, a specific type of female appears which gives birth to a winged generation of males and fe­males, called the fall migrants[7]. The fall mi­grants move back to bird cherry, where they mate and lay eggs. Only these eggs can survive the Swedish winter, thus finishing the aphid life circle[8].

Plant defence reactions

Plants respond to insect assault with induction of various defence reactions. A type of sub­stances, in­volved in defence, is secondary me­tabolites, which subdivide into three main groups: ter­penes, phenolic compounds and ni­tro­gen-con­taining secondary products. In most cases the secondary metabolites are present at non-induced condition, and only some of them are synthesized depending on the circum­stan-ces. A big group of N-containing secon­dary me­tabolites are water-soluble basic toxins, al­ka­loids, synthesized from the amino acids as­partic acid, lysine, tyrosine and tryptophan. Al­kaloids have one or several basic nitrogen atoms, which are often built into a heterocyclic ring system. Al­kaloids have strong physiologi­cal effects in defence against herbi­vores. Some al­kaloids interfere with components of the nerv­ous system, especially chemical trans­mit­ters; other affect mem­brane transport, pro­tein synthesis or miscella­neous enzyme activi­ties[9].

 

A significant roll in plant defence reactions play signal transduction pathways, which am­plify the original signal and result in the acti­vation or repression of genes. Phloem-feeding aphids penetrate with the stylet to the veins between cells, damaging the plant tis­sue only slightly. They are perceived by the plants as patho­gens, what is leading to the induction of the pathogen-defence response pathways, the sali­cylic acid- and jasmonic acid/ethylene-de­pend­ent signalling pathways1.  Jas­monic acid (JA) is a signalling compound in­volved in multi­ple aspects of plant responses to their bi­otic and abiotic environment. Synthe­sised from membrane lipids, JA can induce ex­pression of a range of early and late func­tion­ing defense genes[10]. A barley gene, which was found to be induced by bird cherry-oat aphid and also by jasmonic acid, was identified as an O-methyl­transferase gene[11]. In another recent study, an OMT-gene was found to be in­duced fur­thermore by another aphid species (greenbug, Schizaphis graminum) in sorghum 10.

Gramine

One secondary metabolite, which has been found to be induced in barley upon aphid in­festation, is gramine 12. The indole proto-al­kaloid gramine has high physiological effec­tiveness necessary for anti­herbi­vore defence. Gramine is toxic to aphids in feeding experi­ments with holidic diets, de­creasing aphids’ longevity and fecun­dity. This indole proto-al­kaloid is found in epi­dermis and in mesophyll parenchyma, but it is missing in the vascular bundles. Different fac­tors like pho­toperiod, tem­perature and plant age have an effect on the amount of gramine[12]. It has been re­ported that gramine inhibits the respiratory chain at com­plex I in rat liver mitochondria and in bo­vine heart submitochondrial particles. It was shown that gramine can inhibit the photo­syn­thetic phosphorylation in spinach thylako­ids, and also reduce energy transfer in photo­syn­thetic units of freshwater blue-green algae Anabaena sp.  [13].

 

Gramine has been detected in the grass family Poaceae (see Figure 1), i. a. genus Phalaris spp., Avena, Hordeum, and also in the family Fabaceae genus Lupinus[14].

 

 

 

Figure 1 The Poaceae Family Tree. Hordeum provides barley, Secale – rye, Triticum – wheat, Avena – oats, Leersia – rice, Zea – corn or maize, Sorghum – millet. Gramine has been found in Hordeum, Avena and Phalaris

 

Young plants have a ten­dency to produce more gramine, but later the gramine content dimin­ishes. The vul­nerability of barley to aphids de­creases with the increas­ing gramine amount in the leaves.

 

The gramine synthesis pathway is not com­pletely understood. In earlier studies [15] it was sug­gested, that gramine could be synthesized from tryptophan via 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI) (see Figure 2). An N-methyltrans­ferase (NMT) catalyzing S-adenosylmethionine (SAM)-de­pendent conversion of AMI to MAMI and from MAMI to gramine has previ­ously been identi­fied 15. Two genes control the gramine synthesis, one (Ami) con­ducting the conversion of tryptophan to AMI, the other (Nmt) encoding an NMT enzyme. It has been suggested, that there may be more than two genes de­termining gramine biosynthesis, be­cause the conversion of tryptophan to AMI probably proceeds in several steps 15.

 

 

 

Figure 2 NMT catalyzing SAM-dependent conver­sion of AMI to MAMI and from MAMI to gramine (see more www.hort.purdue.edu)

O-methyltransferase

The OMT gene, which is the focus of this study (OMT; EC2.1.1.6, GeneBank accession U54767), has been characterized as coding for an O-methyltrans­ferase, acting on the phenolic substrate caffeic acid[16]. In bar­ley it is in­duced by the signal sub­stance jas­monic acid, regulating a pathway, which in­creases expres­sion of defence-response genes. OMTs gener­ally methy­late caffeic acid and lead to lignin precursors or various classes of flavonoids, some of which are phytoalexins. But for some OMTs caffeic acid was found to be the least ef­fective substrate[17]. OMTs employ SAM as the methyl donor in the methylation of hy­droxyl groups[18].

 

Earlier studies have shown, that feeding of the bird cherry-oat aphid on barley induces the patho­genesis-related proteins chitinase and β-1,3-glucanase 1. Recently subtracted cDNA li­braries from infested and non-in­fested barley plants have been screened to de­tect other genes which are up- or down-regu­lated in response to aphid attack. One of the up-regulated genes, coded for an enzyme, had previously been characterized as an O-methyltrans­ferase[19]. It was proposed, that this en­zyme was fun-ctioning as caffeic acid O-me­thyltrans­ferase, catalyzing the methylation of caffeic acid to ferulic acid[20].

It has recently been shown that not all barley cultivars had the OMT gene in their ge­nome[21].  The OMT gene was detected in 8 out of 12 barley lines tested (Ph. D. student K. Lars­son, per­sonal communication). In the barley va­rie­ties missing the gene, the indole alkaloid gramine was not found either. In all gramine-containing lines OMT was present. Moreover, presence or absence of the OMT gene was not correlated with the bar­ley resistance to aphids21. These ob­servations have led to the hypothesis that the enzyme O-methyltrans­ferase could be in­volved in gramine bio­syn­thesis.

Aim of the project

Thus the aim of the project is to char­ac­terize the aphid-induced methyltransferase with the pur­pose to:

 

Materials and methods

Growth and treatment of plants

Hordeum vulgare, variety Lina, susceptible to the aphids, was sown in November 2003 and 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 floated in Petry dishes with 45 μM jasmonic acid solution for 24 hours in order to induce the OMT-gene. Plant material was frozen in liquid nitrogen and stored until use at -70oC.

Total RNA isolation

Total RNA was isolated from 100 mg green tis­sue using the Plant RNeasy Mini kit (Qiagen) according to the kit protocol. The frozen tissue was ground in a mortar to a fine powder and mixed with lysis buffer RLT. DNA was di­gested during RNA puri­fication by means of the RNase-Free DNase Set (Qiagen) according to the kit protocol.

RT-PCR

Total RNA from leaves treated with jasmonic acid was reverse transcribed into single-stranded cDNA using the First-Strand Synthe­sis System for RT-PCR (Invitrogen) and the PCR machine UNO II (Biometra). The for­ward primer OMTcloneF (5’-GGT GGT CAT ATG GAC AAG ATT TCA GCA CCT TTC TTT AG-3’) and the re­verse primer OMTcloneR2 (5’-CCC GGG CTA CTT GGT GAA CTC AAG AGC GTA-3’) were applied to amplify the coding region of the OMT gene using a proof-reading thermostable DNA po­lymerase (Phusion – High-Fidelity DNA Po­lymerase, Finnzymes, MJ BioWorks). First strand cDNA served as tem­plate for the PCR reaction. The PCR machine used for the sequence amplifying was PTC-100 Programmable Thermal Con­troller Peltier-Effect Cycling, MJ Research. The temperature program for PCR was: 98oC for 30 s, then 30 cycles of 98oC (10 s), 66oC (30 s), and 72oC (45 s). The quality of the syn­thesized OMT-fragment was controlled by 2% agarose gel electrophoresis. The PCR product was purified us­ing the Nu­cleoTrap Nu­cleic Acid Purification Kit (BD Biosciences) ac­cording to its protocol.

Cloning of the OMT gene into the pTYB12 vector

The plasmid pTYB12, which allows the fusion of the cleavable intein tag to the N-terminus of a target protein, was chosen as a vector (sup­plied as part of the IMPACT-CN Protein Puri­fication System, BioLabs, New England). The plasmid was digested with the restriction nu­cleases SmaI and NdeI (Fer­mentas Life Sci­ences). The DNA fragment was digested with the restriction nuclease NdeI. The digesting re­actions were carried out at 37oC, except for SmaI which was incubated at 30 oC.  The di­gested DNA was ligated into the pTYB12 using the BioLabs Quick Ligation Kit.

Transformation of E. coli DH5α-T1 and screening for recombinants

The new con­struct pTYB12-OMT was used to transform E. coli DH5α-T1 com­petent cells for amplifying of the plasmid ac­cording to the One Shot Chemical Transforma­tion Protocol (Invi­trogen). The re­combinant cells were se­lected on Petri dishes with LB/amp medium (1% tryptone, 0,5% yeast extract, 1% NaCl pH 7,0, 100 μg/ml ampicillin). 96 ran­domly chosen colonies were inoculated in a microtitre plate in LB me­dium with 100 μg/ml ampicillin. Plates were shaken at 37ºC for 1 hour. 1 ml of this culture was used as template for a PCR test for inserts using internal primers OMT F1 (5’-ATA TAG CAG AGG CGG TGA CT-3’) and OMT R1 (5’-AAG AGA ACC GCA TCT CCA GT-3’). PCR conditions were 4 min 94 oC, 35 cy­cles of: 94oC 30 s, 55oC 30s; and 72 oC 1 min (PCR machine used here was PTC-100 Pro­grammable Thermal Controller Peltier-Effect Cycling, MJ Research). Products were ana­lysed by agarose gel electrophoresis. Three clones which gave the expected product for the OMT insert were grown over night in LB me­dium with ampicillin at 37oC with shaking. The plasmid DNA was purified using QIAprep Spin Mini­prep Kit (Qiagen) ac­cording to its proto­col. To confirm the obtained recombinant clones, digesting reac­tions with restriction nu­cleases KpnI, NcoI, NdeI and SapI were car­ried out over night at 37oC. The digest reac­tions were analyzed by electrophoresis on a 1 % aga­rose gel. The gel was run at 100 V for 2 hours.

 

The new construct pTYB12-OMT was con­trolled for the right insert by PCR (PCR ma­chine PC-960G Gradient Thermal Cy­cle) with the three pairs of primers: OMT clone F and OMT clone R2; OMT F1 and OMT R1; and Intein Forward (5’-CCC GCC GCT GCT TTT GCA CGT GAG-3’) and T7 Termi­nator Re­verse (5’-TAT GCT AGT TAT TGC TCA G-3’) and analysed on a 2 % aga­rose gel. The plasmid DNA from the clone con­taining the OMT insert was sent to be se­quenced at Cyber­gene, Novum in Huddinge.

Transformation of the expression strain E. coli ER2566 and screening for recombinants

The E. coli strain ER2566 was pro­vided by Impact-CN Protein Purification Sys­tem (Bio­Labs) as a host strain for the expres­sion of a target gene, cloned in the pTYB12 vector. Competent cells of ER2566 were transformed with the plasmid pTYB12-OMT according to the One Shot Chemical Transfor­mation Proto­col. The same plasmid preparation, which was sent to be sequenced, was used here. The re­combinants were se­lected on Petri dishes with LB medium and 100 μg/ml ampicillin. Colo­nies were picked and grow­n in LB/amp me­dium with shaking at 37o C over night.

Transformation of E. coli ER2566 as the positive control of the induction reaction

To control the protein induction, E. coli ER2566 was transformed with pMYB5 vector, as it was recommended by the instruc­tion man­ual of the IMPACT-CN Protein Purifi­cation System. The transformation was carried out ac­cording to the One Shot Chemical Trans­forma­tion Protocol. The re­combinant cells were se­lected on Petri dishes with LB medium and 100 μg/ml ampicillin. The control clones ER2566-pMYB5 were inoculated in 5 ml LB me­dium with 100 μg/ml ampicillin with shak­ing over night at 37oC and used as positive control for the induction of protein expression.

Induction of protein expression

Different conditions for protein expression were weathered. Expression clones were grown in LB/amp medium (1% tryptone, 0,5% yeast extract, 1% NaCl pH 7,0, 100 μg/ml ampicil­lin). Protein expres­sion was induced with 0,5 mM or 1 mM IPTG (isopropylthiogalacto­side). Tem­perature conditions at 15oC, 20oC and 37oC were tested for the best result. The ex­pression clones were harvested after 4 and 6 hours and also next morning. OD595 was meas­ured spec­tropho­tometrically. The quality of the induction reac­tions was analysed by SDS-PAGE gel electro­phoresis. Two read­y-manu­factured 4-20% Tris-Glycine gels (4-20 % gra­dient Tris Gly­cine gel Cambrex PAGE for polyacryla­mide gel electrophoresis, In Vitro Sweden AB) were loaded with 20 μl of the sam­ples mixed with SDS Sample buffer (final conc. 1×, 50 mM Tris, 2% SDS, 0,1% BPB, 10% glycerol). Samples were boiled in 1×SDS sample buffer at 100oC for 5 min. The gels were run at 125 V and 60 mA for 95 min at room temperature.

Western blot

The total protein was separated using SDS-polyacrylamide gel electrophoresis. The gel was run at 125 V and 30 mA for 100 min at room temperature. The protein bands were trans­ferred from the gel onto Hybond-P PVDF membranes electropho­retically by means of semi-dry transfer appa­ratus at 47 mA during 60 min. Before the incu­bation with primary anti­bodies, the membrane was blocked with blocking so­lution containing 1,25% milk pow­der dissolved in buffer PBS-T (NaCl 8 g/l, KCl 0,2 g/l, Na2HPO4 1,44 g/l, KH2PO4 0,24 g/l, Tween 0,5 ml/l) for 1 hour. The PVDF mem­branes were incubated with the primary anti­body Anti-Chitin Binding Domain Serum di­luted 1:5000 overnight at 4oC. Then the mem­branes were washed with PBS-T three times for 15 min on the shaking table. With the secondary antibody Goat Anti-Rabbit HRP di­luted 1:3000 the membranes were incubated for 2 h at RT. After that the membranes were washed with PBS-T three times for 15 min and with PBS two times for 10 min. The protein was detected using the ECL Plus kit (ECL Plus Western Blotting De­tection Kit, Am­ersham Biosciences) and chemilumines­cence in the CCD-camera.

Purification of the target protein

The methyltransferase was purified using the IMPACT-CN Protein Purification System, BioLabs, New England. 1 liter of cell culture was grown at 37oC until OD595 was 1,23. Pro­tein expression was in­duced with 1 mM IPTG (isopropylthiogalacto­side) at RT overnight. The cells were harvested by cen­trifugation at 5000 rpm for 10 min at 4oC, and resuspended in Cell Lysis Buffer (20 mM Tris-HCl pH 8,0, 500 mM NaCl, 1 mM EDTA, 0,1% Tween 20). Cell lysis was achieved by sonication. The clarified cell ex­tract was ob­tained by centrifu­gation at 17700 rpm for 30 min at 4oC, and then it was parti­tioned and loaded onto three 15 ml chitin col­umns (Poly­propylene disposable columns, 5 ml, QIAGEN GmbH, Hilden). The columns were washed with the Col­umn Buffer (20 mM Tris-HCl pH 8,0, 500 mM NaCl, 1 mM EDTA), and the on-column cleav­age reac­tions were started by adding Cleavage Buffer (20 mM Tris-HCl pH 7,5, 500 mM NaCl, 1 mM EDTA, 50 mM DTT). The cleavage reac­tions were carried out at following conditions: one - at 4oC for 24 h, another – at RT for 24 h and the third – at RT for 40 hours.  The NaCl concentra­tions of 0,5 M and 1 M in the buffers were also tested to achieve the highest effi­ciency of the target protein purifi­cation. The protein was eluted using the Col­umn Buffer. The concen­tration of the obtained protein sam­ples was measured spectropho­tometrically using the Bradford microassay method for protein quan­tification. The effi­ciency of the cleavage reac­tions was analyzed by SDS-PAGE gel electro­phoresis.

Assay of the methyltransferase activity with AMI and MAMI

Purified enzyme was used for assays, based on T. J. Leland’s and A. D. Hanson’s as­say for NMT activity 15, employing chemically synthe­sized (by Ann-Louise Johnson, KI) AMI and MAMI as substrates. Methyl­transferase activity was measured by estimation of the amount of 3H-labelled product produced with methyl-3H-SAM. For determination of the ki­netic pa­rameters of the methyltrans­ferase, the assays were performed for different incubation times (0, 30 and 60 min) and also for different con­centrations of AMI and MAMI (0,75, 1,5 and 3 mM). All re­actions were done in duplicate. The assay mixture contained 55 μl enzyme, 0,15 M Tris-HCl pH 9,0, 1 mM MgCl2, AMI or MAMI of the re­quired concentration, and 0,6 mM SAM + 3H-SAM, 5 nCi/ μl. After 30 and 60 min incu­ba­tion in a shaking water-bath at 30oC, the re­ac­tions were stopped by adding 400 μl of 1 M H3BO3-Na2CO3 pH 10,0. Alkaloids were ex­tracted into 500 μl CHCl3, and 400 μl of the chloroform phase were taken for thin layer chromatography (TLC) analysis. After the chloroform evaporation in the speed vacuum centrifuge, alkaloids were dissolved in 50 μl methanol and applied on the TLC-plates (20×20 cm Silica gel 60, Merck KGaA, Darm­stadt, Germany). The plates were developed standing in TLC solvent (CHCl3 – MeOH – NH4OH conc. 80:15:1 v/v) for 80 min. To visualize the alka­loids separation, the dry TLC-plates were sprayed with Urk-Salkowski detec­tion reagent ({A} Van Urk reagent: 1 g p-di­methylaminobenzaldehyde, 50 ml conc. HCl, 50 ml ethanol; {B} Salkowski reagent: 2.03 g FeCI3 . 6 H2O, 500 ml H2O, 300 ml conc. H2SO4; the TLC spray reagent, was made up of reagent A and B (1:3) [22]). The TLC-plates were heated in the oven at 100oC for 8 min and im­mersed in 3 l distilled water three times. The regions containing the reaction products were scraped from the dry TLC-plates for liquid scintillation counting. 3H count per minute was recalculated into built product, pmol/min per 1 mg protein, using Microsoft Excel. The coeffi­cient Rf for AMI, MAMI and gramine was cal­cu­lated as

,

where a - space between the start line and the built products position; b - space between the start and the solvent front lines on the TLC-plate.

Assay of the methyltransferase activity with caffeic acid

The enzyme activity with caffeic acid was analyzed as described by F. E. Pak17. The assay mixture contained 27,5 μl en­zyme, 0,15 M Tris-HCl pH 9,0, 1 mM MgCl2, 3 mM caf­feic acid, and 0,6 mM SAM + 3H-SAM, 5 nCi/μl. After incubation in a shaking water-bath at 30oC, the reactions were stopped by adding 2,5 μl of 6 M HCl. The me­thylated products were extracted into 100 μl ethyl ace­tate, and 20 μl was taken for liquid scintillation counting. To verify the optimal conditions, the reaction was also performed with Tris-HCl pH 7,5. The methyltransferase extract from green tissue of barley vari­ety Lina was taken as a control for this ex­peri­ment. To investigate the kinetic properties of the enzyme the incu­bation of the samples was stopped after 30 and 60 min.

Results

Cloning of the OMT gene into the pTYB 12 vec­tor

The total RNA, isolated from barley green tis­sue, was converted into single-stranded cDNA by reverse transcription. First strand cDNA served as tem­plate for the PCR reaction. A product of about 1100 bp was visualized by 2% agarose gel electrophore­sis (see Figure 3).

 

 

Figure 3 Agarose gel electrophoresis of the RT-PCR. Well M is loaded with DNA Ladder mix, well 1 - with the RT-PCR

The plasmid pTYB12 and the DNA fragment were digested with restriction nucleases as de­scribed above and ligated to produce the plas­mid pTYB12-OMT.

 

It became obvious that E. coli DH5α-T1 was not easy to transform using the pTYB12-OMT plasmid DNA. After four trials merely the last one was successful, which in the end resulted in only one clone with the correct insert. The re­combinant cells were selected on Petri dishes, and 96 colonies were inoculated in the microti­tre plate in LB me­dium with am­picillin. A PCR test for inserts was done, and the agarose gel electrophoresis showed that three clones con­tained the OMT insert. These clones numbered 1, 2, and 3 were grown over night in LB me­dium with ampicillin, and the plasmid DNA was extracted.

 

The pTYB12-OMT recombinants were further tested by restriction analysis with restriction nucleases KpnI, NcoI, NdeI and SapI. The di­gested DNA was analyzed on 1 % aga­rose gel. This re­vealed that one clone gave the ex­pected fragment pattern and thus was chosen as the pTYB12-OMT plasmid (see Figure 4). Lanes 1 – 4 are corre­sponded to the digesting reactions of the plas­mid DNA 1, lanes 7 – 10 belong to the plasmid DNA 2, and the four last lanes to the plasmid 3. The clone 1 has the ex­pected fragment pattern, which corre­sponds to the pTYB12 plasmid DNA with the in­sert of the OMT-gene coding region. The digesting reac­tion with the restriction nuclease KpnI (see Figure 4, lane 1) has given two bands of the

 

Figure 4 Agarose gel electrophoresis of the re­striction analysis of putative clones with KpnI, NcoI, NdeI and SapI digesting reactions. Lanes 1 – 4 are corresponded to the digesting reactions of the plasmid DNA 1, lanes 7 – 10 belong to the plasmid DNA 2, and the four last lanes - to the plasmid 3. The wells are loaded with the combina­tions of clones and restric­tion enzymes as follows: 1 – clone 1, KpnI; 2 – clone 1, NcoI; 3 – clone 1, NdeI; 4 – clone 1, SapI;  5 – non-digested clone as a control; 6 – DNA Lad­der mix;  7 – clone 2, KpnI; 8 – clone 2, NcoI; 9 – clone 2, NdeI; 10 – clone 2, SapI; 11 – clone 3, KpnI; 12 – clone 3, NcoI; 13 – clone 3, NdeI; 14 – clone 3, SapI

 

size of about 6700 and 1800 bp. The digesting reaction with the restriction nuclease NcoI (see Figure 4, lane 2) resulted in three bands of the sizes 7400, 700 and 450 bp. The digest­ing reaction with the restriction nuclease NdeI (see Figure 4, lane 3) shows a band of about 8500 bp. And finally the digesting reac­tion with the restriction nu­clease SapI (see Figure 4, lane 4) is showing the fragments of 7800 and 700 bp. The clones 2 and 3 have different fragment patterns demon­strating that they do not contain the correct in­sert. One recombinant possessing the pTYB12-OMT plasmid DNA, the clone 1, had been ob­tained and was chosen to continue the ex­peri­ment.

 

The new construct pTYB12-OMT was con­trolled for the right insert by PCR with the three pairs of primers: OMT clone F and OMT clone R2; OMT F1 and OMT R1, and Intein Forward and T7 Termi­nator Reverse. Bands of the correct sizes were visible on 2 % agarose gel (see Figure 5). The product of the PCR with the primers OMT clone F and OMT clone R2 (see Figure 5, lane 1) is about 1100 bp. The PCR with the primers OMT F1 and OMT R1 (see Figure 5, lane 2) has re­sulted in a band of about 350 bp. The PCR with the primers Intein Forward and T7 Terminator Reverse (see Fi­g­ure 5, lane 3) produced a band of about 1300 bp.

 

The PCR reactions have proved that the coding region of the OMT-gene was inserted into the expression vector pTYB12.

 

 

Figure 5 Agarose gel electrophoresis of the PCR reaction. The first well to the left, M is loaded with DNA Ladder Plus. Wells 1 – 3 are loaded with PCR product with following primers­: 1 - OMT clone F and OMT clone R2; 2 - OMT F1 and OMT R1; 3 - Intein Forward and T7 Terminator Reverse.

 

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 19 (see Figure 6).

Transformation of the expression strain E. coli ER2566

Transformed competent cells E. coli ER2566 were se­lected on Petri dishes with LB/amp me­dium. To control the pro­tein induction E. coli ER2566 was also trans­formed using the pMYB5 vector. Quite a num­ber of colonies were obtained on Petri dishes in both cases. The cultures were inoculated for protein induc­tion.

 

Figure 6 Complete insert from pTYB12-OMT. The primers OMTcloneF, OMT F1 and OMTR1 are marked in red. The size of the insert is 1128 bp.

Induction of protein expression

The protein expression was induced with 0,5 mM IPTG (see Figure 7). Bands of a size of about 100 kDa, which corresponds to the ex­pected size of the fusion protein intein-methyl­transferase, could be seen in all the lanes ex­cept the lane M, marker, and lane 2, the non-induced sample. The lanes 3, 7 and 9 show the strongest bands, accounting for the highest synthesis of the fusion protein. Therefore the expression clones E2 and E6 were preferred to continue the trial (see Figure 7, lanes 3 and 7).

 

 

Figure 7 SDS-PAGE analysis of the in­duction re­action. Well M is loaded with Protein Marker Broad Range; the scale is shown in kDa. Wells 1 - 9 are loaded with pro­tein samples as follow: 1 – the positive control clone, ER2566-pMYB5 in­duced in LB medium at 15oC; 2 – non-induced expres­sion clone E1, 3 – ex­pression clone E2, 4 - E3, 5 – E4, 6 - E5, 7 – E6, 8 - E7, 9 –E8

 

To further optimize the protein induction con­ditions, the protein expression was induced with 0,5 mM or 1 mM IPTG. Tem­perature conditions at 15oC, 20oC and 37oC were tested for the most optimal result (see Figure 8). The expression clones grown at 37oC were har­vested after 4 and 6 hours, and the clones grown at 15oC and RT were harvested next morning.

 

Figure 8 Analysis of the different conditions for the induction of protein expression by SDS-PAGE. The gel demonstrates the ex­pression clone E6 in­duction with 0,5 and 1 mM IPTG at 15oC (lanes 9-10), RT (lanes 7-8) and 37 oC (lanes 3-6). The lanes 1 – 10 are corre­sponded to: 1 - non-induced E1 as a negative con­trol; 2 - Protein Marker Broad Range; 3 – induc­tion with 0,5 mM for 4 h; 4 - 1 mM IPTG, 4 h; 5 - 0,5 mM IPTG, 6 h; 6 - 1 mM IPTG, 6 h; 7 - 0,5 mM IPTG; 8 - 1 mM IPTG; 9 - 0,5 mM IPTG; 10 - 0,5 mM IPTG

SDS-PAGE gel analysis showed that the strongest band of about 100 kDa, corre­sponding to the size of the intein-methyltrans­ferase fusion protein, was visible in lane 8, when the protein expression was in­duc­ed with 1 mM IPTG at RT overnight.

Western blot

Total protein extracted from induced E. coli cells was separated using SDS-poly­acrylamide gel electrophoresis, and afterwards the sepa­rated bands were transferred elec­tropho­retically onto PVDF membranes. After the im­munoblotting using antibodies against the chi­tin binding do­main of the fusion protein, the protein was de­tected using the ECL Plus West­ern Blotting kit and chemilumines­cence in the CCD-camera (see Figure 9).

 

Figure 9 Immunoblotting with antibodies against the chitin-binding domain. Lane 1 contains size marker (Kaleidoscope Prestained Standards). Lane 2 shows the posi­tive control clone, ER2566-pMYB5 in­duced in LB me­dium at 15oC. Lanes 3 – 10 are the results of the following samples:  3 – the non-induced ex­pres­sion clone E1 as a negative control; 4 – E2 induced in LB/amp me­dium at RT with 0,5 mM IPTG; 5 – E2 induced in LB/amp me­dium at RT with 1 mM IPTG; 6 – E6 induced in LB/amp me­dium at RT with 0,5 mM IPTG; 7 – E6 induced in LB/amp me­dium at RT with 1 mM IPTG; 8 – E8 in­duced with 0,5 mM IPTG; 9 – expression clone E12 induced with 0,5 mM IPTG; 10 - expres­sion clone E16 in­duced with 0,5 mM IPTG

 

The western blotting showed the strongest bands of about 100 kDa in lanes 4 – 7 and has proved that the induction of the protein expres­sion was successful. The expression clone E6 induced in LB/amp medium at RT with 1 mM IPTG has showed the strongest band, which means that these conditions were the best for the protein expression.

Purification of the target protein

The target protein was purified using the IM­PACT-CN Protein Purification System. 1 l cell culture was grown at 37oC until OD595 was 1,23. The expression cells were induced with 1 mM IPTG at RT overnight. The OD595 of the harvested cells was 1,995. The cells were bro­ken by sonication, and 50 ml clarified cell ex­tract, obtained by centrifugation, was par­ti­tioned and loaded onto three chitin columns. Different conditions for the on-column cleav­age reaction were tested: at 4oC and RT for 24 and 40 hours. The highest protein concentra­tion was obtained by elution with the Column Buffer containing 0,5 M NaCl. 6 ml eluant 1, 6 ml eluant 2 and also 6 ml eluant 3 were ac­quired for each of the three cleavage reactions. The protein concentration of the samples was measured spectrophotometrically, using Brad­ford mi­croas­say method for protein quantifica­tion (see Table 1).

 

Table 1 Concentration of the target protein

Cleavage reaction conditions

Concentration of the target protein, μg/ml

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

 

The efficiency of the protein purification was analyzed by SDS-PAGE gel electrophoresis (see Figure 10). The strong bands of about 100 kDa, corresponding to the fusion protein intein-methyltransferase, can be seen in the lanes 3 and 4 of the samples before loading onto the chitin column. Lane 4 is illustrating the flow-through taken after the chitin column was loaded with the clarified cell extract. The pale band of the size 100 kDa proves that the fusion protein intein-methyltransferase has bound to the chitin beads. The lanes 5 and 6 do not have any bands at all, as expected for the column and DTT wash. The wells 7 - 9 were loaded with the 1 - 3 fractions respectively of the eluant 1. The bands of about 43 kDa corre­spond­ing to methyltrans­ferase are clearly visi­ble. The amount of the protein in the eluant 2 was insig­nificant and could not be detected by SDS-PAGE gel elec­trophoresis. The cleavage re­ac­tion at 4oC was ineffective and the small amount of the protein could not be detected by SDS-PAGE gel analysis either. Totally 3,138 mg methyltransferase from 1 l cell culture were ob­tained by the IM­PACT-CN Protein Purifica­tion System.

 

 

Figure 10 Efficiency of the purification of the tar­get protein using buffers with 0,5 M NaCl, the cleavage reaction conditions – 24 h at RT. The lane M is corre­sponding to the Protein Marker Broad Range. Lane 1 shows the crude extract from unin­duced cells.  The lanes 2 – 9 are results of the fol­lowing samples:  2 – the crude extract from the in­duced cell culture E6; 3 – the clarified cell ex­tract, 4 – the chitin column flow through; 5 – the chitin column wash; 6 -  quick DTT wash; 7 - 9 fractions 1 – 3 respectively of the eluant 1 of the cleavage reaction

Assay of the methyltransferase activity

For determination of the kinetic parameters of the methyltrans­ferase, AMI, MAMI and caffeic acid were used as substrates. The AMI and MAMI products were separated by means of TLC-plates (see Figure 11). The regions with the reaction products were scraped from the TLC-plates for liq­uid scintillation counting. 3H count per minute was calculated into built product per 1 mg protein (see Table 2)

 

Table 2 AMI and MAMI methylation products built per 1 mg protein, pmol/min, development in time

Incubation time, min

0 min

30 min

60 min

AMI

0

226,3±4,6

148±2,4

MAMI

0

52,6±7,9

71,2±9,8

Lina, AMI

0

-

16,6

 

As it is seen from the Table 2, the methylation of AMI is highest after 30 min incubation, and it decreases later.  The methy­lation of MAMI increases in time and is high­est after 60 min in­cubation. The enzymatic activity was higher with AMI as substrate than with MAMI.

 

 

Figure 11 TLC analysis of the methyltransferase activity with AMI as substrate. The separation of the methylation products is shown by the arrow. The lanes 1 and 7 are corresponded to AMI stan­dard, the lane 2 – to MAMI standard. the remain­der illustrate following samples: 3 and 4 – product at 0 min; 5 and 6 – reactions with 3 mM AMI for 30 min; 8 and 9 – 1,5 mM 30 min; 10 and 11 – 0,75 mM 30 min.

 

The methyltrans­ferase activity was analyzed relatively the AMI and MAMI concentration (see Table 3). MAMI built from AMI is in­versely proportional to the substrate concentra­tion. The methylation of MAMI has a different pattern, and the experiment data can be ques­tioned.

 

Table 3 AMI and MAMI methylation products built per 1 mg protein, pmol/min, relative to the substrates concentration

Concentration, mM

0,75 mM

1,5 mM

3 mM

AMI

37,03±10,2

18,71±1,4

21,34±4,7

MAMI

2,38±0,9

-

1,53±0,9

 

The coefficient Rf for AMI, MAMI and gramine was calculated (see Table 4). Rf be­longs to the interval [0.15; 0.35] and an align­ment between its value is as follow:

 

RfMAMI < RfAMI < RfGramine

 

Table 4 Coefficient Rf for AMI, MAMI and Gramine

Substrate

Rf

AMI

0,27

MAMI

0,15

Gramine

0,35

 

The enzyme activity with caffeic acid was also analyzed. The reactions with the enzyme extract from barley (variety Lina) green tissue did not show any activity. The re­actions with the me­thyltransferase purified by IM­PACT-CN Pro­tein Purification System ob­tained some built product, but the data are questionable because of the experimental problems.

 

Discussion

Cloning of the OMT gene into the pTYB12 vector

The purpose of this study was to express the aphid-induced barley me­thyltransferase in Es­cherichia coli and to characterize the en­zyme. With this point of view 5-days-old bar­ley plants green tissue was treated with jasmonic acid (JA) to induce the OMT-gene. JA is serv­ing as the intracellular defense signal, mediat­ing expression of a range of early and late func­tioning defense genes 9. It had been pub­lished earlier that also the OMT-gene is induced by JA20. The plasmid pTYB12 in­cluded in IMPACT Protein Purification System was chosen as a vector. A reason for that was that pTYB12 contained suitable restriction sites, which enable the fu­sion of the cleavable intein tag to the N-termi­nus of the target me­thyltrans­ferase[23]. The plasmid pTYB12 was digested with the re­stric­tion nucleases Sma I and NdeI. SmaI recog­nizes the sequence 5’…CCC GGG…3’ di­gesting between C and G, and its cleavage leaves blunt ends. The cleavage with NdeI is staggered and creates co­hesive ends. The PCR product was digested only with NdeI to create compatible 5’ ends for cloning. The 3’end is al­ready blunt ended after the PCR with Phusion DNA po­lymerase. This enables its ligation with the plasmid DNA. The new plasmid pTYB12-OMT with the size of 8545 bp was obtained after the liga­tion re­ac­tion.

Transformation of E. coli and screening for recombinants

With purpose to amplify pTYB12-OMT, E. coli DH5α-T1 competent cells were trans­formed using the new plasmid DNA. The re­combinant cells were selected employing the recombinant clones ability of resistance to the an­tibiotic ampicillin. Only the cells which had taken up the plasmid pTYB12-OMT could sur­vive and grow on the plates with ampicillin. Thus ran­domly chosen colonies were in­ocu­lated in LB/amp medium, and plasmid DNA was obtained by purification.

 

Several attempts to acquire the positive recom­binants had failed. It was already experienced in earlier studies 20 that 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. In this pro­ject the third transformation resulted in a frameshift mutation – deletion of two base pairs with following stop codon leading to a change in reading frame. Eventually the fourth round of transforming E. coli DH5α-T1 was suc­cessful.

 

To confirm the pTYB12-OMT recombinants, digesting reactions with restriction nucle­ases KpnI, NcoI, NdeI and SapI were per­formed. The result of the digesting reactions was ana­lyzed by 1 % aga­rose gel electrophore­sis (see Figure 4). It is known that the cutting of the plasmid with insert produces the fol­lowing fragments: by the restriction nucle­ase KpnI - 6706 and 1801 bp; NcoI - 7380, 680 and 447 bp; NdeI - 8507 bp; and SapI - 7810 and 697 bp. The test of the three putative clones, which gave the expected PCR product during the in­sert ampli­fication, re­vealed only one clone with the correct restriction fragment pattern. This once more indicates that E. coli, containing OMT, forms mu­tants to survive. The fact, that the clone ob­tained for pTYB12-OMT, had the correct se­quence with­out any mutation, was fi­nally con­firmed by se­quence analysis of the in­sert. This analysis is especially important for the reason, that the protein, purified from E. coli for the enzyme measurements, must have the cor­rect amino acid sequence.

Transformation of the expression strain E. coli ER2566 and induction of protein expression

Competent cells E. coli ER2566 were provided by the IM­PACT-CN Protein Purifi­cation Sys­tem as a host strain for the expres­sion of a tar­get gene cloned into the pTYB12 vector. ER2566 cells 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 iso­propylthio­galacto­side (IPTG), by the binding of lac I rep­ressor to the lac promoter. When IPTG is ac­cessible, the transcription of the T7 polymerase gene is induced. The T7 RNA po­lymerase will then start transcription of the RNA for the fusion protein, which is under control of the T7 promoter.

 

The protein expression was induced with 0,5 mM IPTG (see Figure 7). SDS-PAGE analysis showed that this led to the induction of a strong protein band of about 100 kDa, which is in agreement with the expected size of the fusion protein intein-me­thyltransferase.

 

Among the different conditions, investigated for the induction of the protein, 20oC over night and 1 mM IPTG were the most optimal. SDS-PAGE gel analysis has obtained the strongest band, which corre­sponded to the size of the intein-methyl­trans­ferase fusion protein, for these conditions (see Figure 8). Western blot­ting has confirmed this result.

Purification of the target protein

The chitin-binding domain (CBD) in the intein tag allows purification of the fusion protein using a chitin column. The CBD has very high affinity for the chitin beads, which allows ef­fective recovery of the fusion protein from the crude cell extract. The target protein was puri­fied, and SDS-PAGE analysis showed a major band at 43 kDa in the eluant after the on-col­umn cleavage reaction (see Figure 9). As it was expected the strong bands about 100 kDa, cor­responding to the induced fusion protein intein-methyltransferase, can be seen at the lanes 3 and 4 of the samples before the loading onto the chitin column. It has been described previ­ously that in an attempt to express the OMT protein in E. coli most of the protein was lo­cated in inclusion bodies 18. Also in this ex­periment the 100 kDa fusion protein band was stronger in the crude cell extract as compared to the clarified lysate. This could be explained that some part of the protein in the E. coli cells was in the non-soluble fraction. The flow-through (lane 4) taken after the chitin column was loaded with the clarified cell extract, shows only a pale band of the size 100 kDa, confirm­ing that the fusion pro­tein intein-me­thyltrans­ferase has bound to the chitin beads. The column and DTT wash (lanes 5 and 6 re­spec­tively) do not have any bands at all, proving the high effi­ciency and specificity of the chitin beads bind­ing capacity. The bands about 43 kDa corre­spond­ing to the methyltrans­ferase were ob­tained at lanes 7 - 9, corresponded to the 1 - 3 fractions respectively of the eluant 1. In addi­tion, higher salt concentration, 1 M NaCl, was employed to reduce non-specific binding, thus increasing purity. But the higher salt concen­tration did reduce the purified pro­tein concen­tration. The cleavage reaction at room temperature car­ried out for 24 h and using buffers contain­ing 0,5 M NaCl was the most effectual resulting in the pure protein.

Assay of the methyltransferase activity

Purified protein was used in enzyme assays with intermediates in gramine biosynthesis pathway, AMI (3-ami­nomethylindole) and MAMI (N-methyl-3-ami­nomethylindole), as substrate, as well as with caffeic acid, because the enzyme was previously described as caffeic acid OMT.  AMI and MAMI were both acting as substrates, and transformed to MAMI and gramine, respec­tively. 3H count per minute was recalculated to build product, pmol/min, per 1 mg protein (see Table 2). The methylation of AMI was highest after 30 min incubation, de­creasing later, which contradicts the kinetic de­velopment in time as a logarithmic function.  The methy­lation of MAMI increases in time and is high­est after 60 min in­cubation. The methyltrans­ferase activity was ana­lyzed relatively to AMI and MAMI concentra­tion. The production of MAMI from AMI was in­versely propor­tional to the substrate concentra­tion, indicating product inhibition.

 

The experiment has revealed that the coeffi­cient Rf for AMI, MAMI and gramine, calcu­lated as a dividend of the interval between the start line - built products and the start - solvent front lines on the TLC-plate, is not constant. It varies for different TLC-plates and seems to be dependent on the silica gel layer quality and the TLC solvent composition. It can be reasonable to apply the substrates samples onto each TLC-plate as a control in further tests.

 

The experiments, measuring the enzyme ac­tivity with caffeic acid as sub­strate, were diffi­cult to interpret because of the experimental problems. There seems to be very little ac­tivity, but the assay must be developed to achieve some reliable results. It has to be noted that 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. Thus these experi­ments have to be repeated with freshly purified enzyme. However, a freshly extracted protein from green tissue of the barley variety Lina (a variety which contains the OMT gene and has a high gramine content), did not exhibit any ac­tivity with caffeic acid, but did methylate AMI and MAMI. This supports the preliminary re­sults from the studies with the in vitro ex­pressed OMT, indicating that it might be in­volved in gramine synthesis by methylating AMI and MAMI, rather than acting as caffeic acid OMT.

 

The enzyme of interest has been described as an O-me­thyltransferase, and a sequence com­parison with other sequences in data bases in­dicates, that it has similarity to other OMTs. But its maximum similarity is only 40% 20. Thus it could well be, that it 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 gramine.

 

In general terms, this work strongly supports the idea that the methyl­transferase gene acces­sion num­ber U54767, could be involved in gramine bio­synthesis, possibly acting as an NMT.

Acknowledgements

I would like to express my thanks to Ph.D. Professor in Botany Lisbeth Jonsson for her advices for the me­thyltransferase assay performance and her con­siderable comments; to Dr. Gabriele Delp for her help with transformation of E. coli DH5 -T1 and her support during the work; to Ph. D. Student Kristina Larsson for the introduction and support at the lab in the beginning of the project; to Ann-Louise Johnson for the kind donation of the chemically synthesized sub­strates AMI and MAMI; to Ph. D. Student Maria Schubert for her help with the spectrophotometer and Brad­ford assay for protein quantification; to Ph. D. Student Galyna Bartish for her assistance with the CCD-camera and her friendly support dur­ing the project; to Ph. D. Student Rose-Marie Jenvert for her help with the scintillation counter; and to Ph. D. Student Evgeni Oni-schenko for his assistance with the sonica­tor.

 

References



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