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«by CHIEH-TING WANG (Under the Direction of Jeffrey F. D. Dean) ABSTRACT Laccase and related laccase-like multicopper oxidases (LMCOs) have been ...»

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5’- and 3’- RACE 5’-RACE was used to obtain the 5’ untranslated region (UTR) of the At2g30210 gene. In brief, 5µg total RNA isolated from seedling roots was reverse transcribed using the gene-specific reverse primer (1694R) and Superscript II as previously described. The resultant cDNA was used as template for PCR employing the proof-reading polymerase (Promega), in combination with the secondary reverse primer (1061R) and an abridged anchor primer provided by the RACE kit manufacturer (Invitrogen). The PCR product was subsequently cloned into the pCR2.1 vector and sequenced. The potential secondary structure of the 5’UTR was predicted using the RNAfold program at a web interface server (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) (Hofacker et al. 1994).

To capture the sequence of the 3’UTR of the At2g30210 gene, 3’ RACE was performed as described above, and the forward primer (76F) and reverse primer provided by the manufacturer (AUAP),

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into the pCR2.1 vector and sequenced as described previously.

Expression of At2g30210 in E. coli The pTrcHis XpressTM Protein Expression System (Invitrogen) was used to express the LMCO protein in E. coli. The At2g30210 cDNAs, one with the full-length coding sequence (1F) and the other lacking the signal peptide (76F), were subcloned from the cloning vectors into the pTrcHis expression vector digested with the EcoRI restriction enzyme. The resultant constructs were used to transform E. coli Top10 (Invitrogen) or BL21-CondonPlus RP (Stratagene, La Jolla, CA) cells. Correct orientation of the At2g30210 cDNA inserts with respect to the Trc promoter was confirmed by restriction analysis and sequencing. For expression, 50 ml of SOB medium (20g tryptone, 5g yeast extract,

0.5g NaCl, 0.186g KCl in 1 liter water, pH 7.0) containing ampicillin (50µg/ml) was inoculated with 0.3ml of an overnight culture, and incubated at 37 oC with vigorous shaking to reach OD600=0.6. Inducing agent, IPTG, was added to a final concentration of 1 mM, and cultures were continued for 4 hours.

Cells were harvested by centrifugation at 6000 x g for 5 minutes and lysed in using the freeze-and-thaw method described in the Xpress Purification manual (Invitrogen).

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Protein Purification (Invitrogen). In brief, cells were resuspended in 10mL of guanidine lysis buffer (6M guanidine, 20mM sodium phosphate, 500mM NaCl, pH 7.8), and slowly rocked for 10 minutes at room temperature, followed by sonication on ice with three 5-second pulses at high intensity. The insoluble debris was removed by centrifugation at 3000 x g for 15 minutes, and the supernatant was loaded onto a ProBondTM resin column. The column was washed with a series of denaturing washing buffers (8M urea, 20mM sodium phosphate, 500mm sodium chloride) at various pHs (7.8, 6.0, 5.3), and eluted with the denaturing washing buffer at pH 4.0. The eluted fractions were collected in

1.5 ml tubes and aliquots (10µl) were loaded onto gels for SDSPAGE and immunoblot analysis.

Expression of At2g30210 in tobacco cells The cloned full-length coding region (1F) was excised from PCR2.1 in an XbaI/SpeI digest and inserted into XbaI-digested pKYLX80 vector under the control of a dual-enhanced CAMV 35S promoter. Orientation was confirmed by restriction analysis and sequencing. The resultant construct was precipitated onto gold particles (1 µm) and introduced into BY2 cells using a PDSHe Biolistic Particle Delivery System (Bio-Rad, Hercules, CA) equipped with 11 Mpa rupture disks according to the manufacturer’s instructions. Four-day old cultured tobacco cells

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LaFayette at al. (1999). Transformed cells were selected on solid MS medium containing 200mg/L kanamycin and then transferred to new plates containing the chromogenic laccase substrate, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) to screen for phenoloxidase activity.

Cell lines positive for ABTS oxidation were maintained on solid medium containing kanamycin. After several transfers, cells were harvested and lysed in buffer containing 50 mM NaOAc (pH 6.5), 1 mM phenylmethethylsulfonyl fluoride (PMSF), 10 mM NaCl, 1 mM CuSO4 and 10 % glycerol using an OMNI Macro homogenizer (Omni International, Inc., Gainesville, VA). Cell debris was removed by centrifugation at 20,000 x g and 4˚C for 30 min, and supernatant was filtered through Miracloth (Calbiochem-Novabiochem Corp., San Diego, CA). Supernatants were tested directly for phenoloxidase activity without further purification. To test for ferroxidase activity, the supernatant was adjusted to 50% saturation ammonium sulfate and stirred overnight at 4 oC. Insoluble material was removed by centrifugation at 20,000 x g and 4 oC for 30 min, and supernatant was filtered through Miracloth. The resultant enzyme pool was desalted and concentrated by ultrafiltration against a 30 kDa cutoff membrane (Amicon, Beverly, MA), and stored at -80 oC until use.

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Protein was quantified using the Bio-Rad protein assay reagent (Bio-Rad) against bovine serum albumin (BSA) standards.

Phenoloxidase activity was determined from the increase in absorbance at 420 nm due to the oxidation of 5 mM ABTS in 100 mM sodium acetate buffer (pH 5.0) (Hoopes and Dean 2004).

Ferroxidase activity was assayed using ferrous sulfate (100 µM) as the electron donor in 100 mM sodium acetate buffer (pH 5.0) and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine) as a specific chelator to bind ferrous iron remaining at the end of the reaction (De Silva et al. 1997). The assay was also run in a modified reaction using 1µM apotranferrin as Fe3+ acceptor to promote reactions. Protein boiled for 20 minutes was used as a negative control. Reactions were carried out in disposable cuvettes containing 100µM ferrous sulfate in 100 mM sodium acetate buffer (pH 5.0). The reactions were quenched by addition of ferrozine to a final concentration of 1.5 mM after incubation at room temperature. The rate of Fe2+ oxidation was calculated from the decreased absorbance at 560 nm.





Gel electrophoresis SDS-PAGE was performed according to the protocol of Laemmli (1970). For phenoloxidase activity zymogram, crude extracts were mixed 1/1 (v/v) with 2x Laemmli sample buffer containing no

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denaturation. After electrophoresis, gels were stained for phenoloxidase activity using 1,8-diaminonapthalene (DAN) as described previously (Hoopes and Dean 2001). For ferroxidase zymograms, concentrated and semi-purified protein was tested as described (Hoopes and Dean 2004) using the E. coli yacK ferroxidase as a positive control (Kim et al. 2001).

Results Cloning and characterization of the At2g30210 gene from A.

thaliana Although the At2g30210 gene was predicted from genomic sequence information deposited in GenBank, and available from The Arabidopsis Information Resources (TAIR) Database, no fulllength cDNA was available to confirm the predicted gene. To clone the full-length cDNA, a PCR strategy relying on primers based on the genomic sequence was applied. A full-length coding region cDNA (primer 1F, 1714 bp product) and a second cDNA (primer 76F, 1639bp product) lacking the predicted signal peptide were amplified and cloned, respectively, from cDNA pools derived from 14-day old seedling root tissues. In addition, four different lengths of 3’ untranslated region (3’UTR), ranging from 179 to 230 bps, with the consensus AAUAAA sequence and poly(A)+ tail were also cloned. A search of the Arabidopsis EST

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containing a putative 5’ untranslated region (5’UTR), although that 5’UTR was not used in predicting the At2g30210 gene model.

To clone the 5’UTR and confirm the transcription initiation site, a 5’-RACE protocol was used to recover an additional 47 bases of sequence upstream of the translation initiation codon.

Comparison of sequence data for the cDNAs with genomic DNA allowed us to generate a gene structure (Figure 2.2), which agreed with the predicted gene model, except for the 47 bp UTR upstream of the translation initiation codon. In the 5’UTR region, an (AG)3 repeat flanked the 5’ side of the translation initiation codon. Various lengths of AG repeats were also reported in the 5’UTRs of four different LMCO genes isolated from Liriodendron tulipifera. In addition, the predicted secondary structure (Figure 2.3) of this 5’UTR suggested two hairpin structures, similar to iron-response elements (IREs) found in ferritin and transferrin receptor genes in animals.

This data suggested possible regulation of the At2g30210 LMCO at the post-transcription level.

Amino acid analysis showed that At2g30210p contains 570 amino acids with a predicted molecular mass of 64 kDa, and possesses the conserved copper binding domains that characterize multicopper oxidases. The Amino acid sequence also contains a putative glycosyl hydrolase family 1 signature near the carboxyl

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point at pH 9.67 and harbors 7 potential N-linked glycosylation sites. Analysis of the inferred protein sequence using SignalP indicated that At2g30210p has a functional signal sequence and may be targeted to the secretion pathway. Furthermore, the Nterminus of the signal peptide contained twin arginine residues, a feature that could possibly target At2g30210p to the TAT (Twin Arginine Translocation) protein transport system, which has been reported to transport folded proteins across membranes in bacteria and plastids (Cristobal et al. 1999, Berks et al.

2000).

Heterologous expression of the At2g30210 gene product in E. coli Previous studies showed that at least eight Arabidopsis LMCO genes are simultaneously expressed in young root tissues (McCaig et al. 2005). To obtain large quantities of individual proteins for enzymatic activity analyses, the recombinant At2g30210 proteins were expressed first in E. coli. The full-length cDNAs with (1F) and without signal peptides (76F) were placed in constructs having a 6x His-tag at the N-terminus, and these were expressed under control of the E. coli Trc promoter. The E.

coli strain, TOP10, was used first for expression studies, but low levels of expression suggested that codon usage differences between E. coli and Arabidopsis might be a problem (Table 2.1).

To improve expression, another strain, BL21-CondonPlus RP

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constructs. This E. coli strain contains extra copies of the argU and proL genes, which encode tRNAs that recognize the arginine codons AGA and AGG and the proline codon CCC, respectively. These are low usage codons in prokaryotes. After screening for positive colonies, recombinant protein was detected by western blot in cell lines carrying the shortened construct (76F) (Figure 2.4), but no expressed enzyme could be detected for the full-length construct (1F). Further analysis showed, however, that the recombinant protein accumulated in insoluble aggregates so that purification could only be accomplished under denaturing conditions. Unfortunately, we were unable to detect any phenoloxidase activity using the laccase substrates, ABTS or 1,8-diaminoaphthalene (DAN). We manipulated various expression conditions to obtain active enzyme, including culturing at low temperature, adding copper, and changing inducer (ITPG) concentration, but no active product was recovered.

Heterologous expression of the At2g30210 gene product in tobacco BY2 cells To overcome the difficulties encountered with the E. coli expression system, a tobacco (BY2) cell-suspension culture system was used to express the At2g31020 gene. The full-length coding region (1F) of the At2g30210 cDNA was inserted into a

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enhanced CaMV 35S promoter. Tobacco cells were transformed with this vector using particle bombardment. Laccase activity was demonstrated in three cell lines identified from amongst 56 kanamycin-resistant tobacco calli by testing the ability of cells to oxidize the laccase substrate, ABTS (Figure 2.5A). No freely soluble laccase activity was detected in the culture medium, as was previously seen in transformed tobacco cells expressing the sycamore maple LMCO gene (Dean et al. 1998).

Proteins extracted from calli of the three resistant cell lines were compared with a control cell line transformed with the pKYLX80 vector. One of these three lines (Fig 2.5A, #2) exhibited a novel H2O2-independent phenoloxidase activity using an in-gel detection method with 1,8-diaminonaphthalene (DAN), a substrate specific for laccase and peroxidase (Hoopes and Dean 2001). Endogenous phenoloxidases were also detected as bands of activity in lanes containing extracts from control BY2 cells (Figure 2.5B). The apparent molecular mass of the recombinant LMCO product was ~64 kDa, as estimated by SDS-PAGE.

Phenoloxidase activity of the cell line was also measured in a liquid assay using ABTS as substrate, and more than 3-fold greater activity was detected in the highest expressing kanamycin-resistant line compared to control cell lines (data not shown). The growth rate of cell lines expressing the

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transformed with empty vector (data not shown). The same observation was previously reported for tobacco cells expressing a LMCO gene from yellow-poplar (Hoopes and Dean 2004).

Recently, ferroxidase activity associated with a plant LMCO gene was demonstrated in vitro (Hoopes and Dean 2004), and similar activity has been seen with LMCOs from other organisms (Kim et al. 1999; Herbik et al. 2002). To test whether the At2g30210 LMCO harbored ferroxidase activity, cell lysates were partially purified by ammonium sulfate precipitation and ultrafiltration. Three independent tests for ferroxidase activity were conducted using a zymogram approach, but no such activity was detected (Figure 2.6A). In addition, a liquid assay using ferrous sulfate as the electron donor also showed no detectable ferroxidase activity for the At2g30210 protein, as judged in comparison to extracted protein from control and transgenic tobacco cell lines (Figure 2.6B).



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