CAAGACATGCTTGAGAT 3,
and 5
AAGTTCATACTTCTAAC 3, respectively. A total of 5 mg of total RNA from different
soybean tissues was used for synthesizing the first strand of cDNA using SUPERSCRIPT™ II
Rnase H
−
REVERSE TRANSCRIPTASE as sug- gested by the manufacturer GIBCOBRL. RT-
PCR conditions were the same as those in 3 RACE except that the annealing temperature for
GmEpb
2
was 45°C. A total of 20 ml of PCR products was electrophoresed on a 1 wv
agarose gel and visualized with ethidium bromide.
2
.
4
. In 6itro folding of bacterially expressed GmEPa
1
and GmEPb
1
proteins The open reading frames including the 5 leader
sequences of GmEPa
1
and GmEPb
1
were PCR- amplified and cloned into the pET-34b + expres-
sion vector
Novagen. The
primers complementary to the lower strands were designed
with a BamH I site at the 5 ends and the primers complementary to the upper strands with a Xho I
site GmEPa
1
: 5 GACGGATCCATGGGAAG- CAACTTGAGGTTTTTG
3, 5
GACCTC- GAGTTAGCTATTTATAAATGCACAATG 3;
GmEPb
1
: 5
GACGGATCCATGGCTGT- CATGGGTGCATTCTTG 3, 5 ACCTCGAG-
TAATTCTGCAGCCCTTCTTTCCTCCTG 3.
PCR products were digested with BamH I and Xho I and ligated to pET-34b + digested with
the same two enzymes. The constructs were then transformed into BL21 DE3 competent E. coli
cells.
An overnight culture of 5 ml of BL21 was inoculated into a 100-ml culture containing 50
m gml kanamycin. The inoculated culture was
grown at 37°C with vigorous shaking until an OD
600
of 1.0. The 100-ml culture was then split into 2 × 50-ml cultures. IPTG was added to one of
the 50-ml cultures to a final concentration of 1 mM. The other culture was used as an uninduced
control. The 2 × 50-ml cultures were further grown for 5 h after IPTG induction. Different
temperatures and media additives were used dur- ing the bacterial growth. Isolation of total cell
proteins, inclusion bodies and cellulose binding domain CBD fused peroxidase were performed
according to the manufacturer Novagen. Inclu- sion bodies were washed twice with buffer 200
mM Tris – Cl pH 8.0, 100 mM EDTA, 100 mM DTT, 10 vv Triton X-100 before being fully
solubilized in 10 vol. of 6 M urea, 1 mM DTT in 50 mM Tris – Cl pH 8.0.
A single-step dilution was used for the dena- tured protein refolding. A total of 10 mg 1mgml
of the inclusion body prep was slowly diluted in 190 ml of PBS, which once diluted, contained 2 M
urea, 5 mM CaCl
2
, 10 mM hemin and 0.1 mM DTT PBS: 137 mM NaCl, 1.47 mM KH
2
PO
4
, 8.10 mM Na
2
HPO
4
, and 2.68 mM KCl, pH 8.0. After overnight incubation at room temperature,
20-ml aliquots of the folding mixtures were trans- ferred to the wells of a microtiter plate, and perox-
idase activity was monitored using substrate tetramethylbenzadine as described by Vierling and
Wilcox [9].
3. Results
3
.
1
. Isolation of soybean peroxidase cDNA The vast number of plant peroxidase sequences
documented and the rapid amplification of cDNA ends RACE technique made possible the genera-
tion of a plant peroxidase-specific probe. A degen- erate
plant peroxidase-specific
primer PSP
corresponding to a highly conserved region, distal heme ligand HFHDCFV was synthesized. Using
PSP and anchor primer complementary to the polydT end of the cDNA, the 3 RACE experi-
ment resulted in amplification of a major DNA band of 900 bp data not shown. The fragment
was cloned and one of the clones was used as probe to screen the cDNA library.
Approximately 2 × 10
5
recombinant phages
from the soybean seedbud cDNA primary library were screened using the plant peroxidase-specific
probe. A total of 25 clones were obtained by primary screening, and 11 positive clones were
recovered after two rounds of PCR using PSP and T
7
vector primers. The four longest clones, desig- nated GmEPa
1
, GmEPa
2
, GmEPb
1
and GmEPb
2
, were further analyzed.
3
.
2
. Nucleotide and deduced protein sequences of the soybean peroxidase cDNA
GmEPa
1
, GmEPa
2
, GmEPb
1
, and GmEPb
2
have been registered in the EMBL Nucleotide Database under the accession numbers U51191,
U51192, U51193 and U51194. They contained 1298, 1326, 1171 and 1145, nucleotides, excluding
polyA tail, with 86, 82, 59 and 38-bp 5 untrans- lated leaders, and 240, 272, 173 and 168-bp 3
untranslated regions, respectively. Two copies of the putative polyadenylation signals AATAAG
are present at nucleotides 25 and 81 upstream of the polyA tail in GmEpa
1
, and 45 and 112 bases upstream of the polyA tail in GmEpa
2
. There was only one copy of the putative polyadenylation
signal AATAAA 42 bases upstream of the polyA tail in GmEpb
1
and 20 bases upstream in GmEpb
2
. The open reading frames ORFs of GmEPa
1
, GmEPa
2
, GmEPb
1
and GmEPb
2
were 972, 972, 942 and 942 bp long. The deduced amino acid
sequences encoded by the four ORFs are shown in Fig. 1. It was predicted from these sequences that
the proteins were synthesized as preproteins of 324, 324, 314 and 314 amino acids with hydropho-
bic putative signal sequences of 21, 21, 22 and 22 residues, respectively. The mature proteins from
GmEPa
1
, GmEPa
2
, GmEPb
1
and GmEPb
2
were designated as a1, a2, b1 and b2. Cleavage of
putative signal sequences releases mature proteins of 303, 303, 292, and 292 residues with theoretical
M
r
of 33 333, 33 333, 32 412 and 32 412 Da. The theoretical pIs of mature a
1
, a
2
, b
1
and b
2
were 6.96, 7.41, 10.04 and 9.05, respectively. There were
six putative glycosylation sites specified by N-X-T S at amino acid residues 56, 69, 128, 142, 183 and
214 in a
1
and a
2
, and four putative glycosylation sites at residues 70, 142, 185 and 195 in b1 and b2.
Peroxidases a
1
and a
2
had the [Q L X X X F Y] motif at the NH
2
terminus that was a feature
Fig. 1. Optimum alignment of amino acid sequences deduced from GmEPa
1
, GmEPa
2
, GmEPb
1
and GmEPb
2
. The 5 leader sequences are in bold italics.
Fig. 2. A Northern blot analysis of GmEPa
1
expression in root R, seedpod Sp, stem S, leaf L, and developing seed
coat Ds. B Ethidium bromide stained RNA gel indicating roughly equal loading of total RNA. C Ethidium bromide
stained RT-PCR products amplified from cDNA synthesized from total RNA of R, Sp, S, L, and Ds. The two primers
were PSP and GmEPa
1
-specific primer. D Southern blot analysis of RT-PCR products amplified using PSP and
GmEPa
1
-specific primers.
The probe
was
32
P-labelled GmEPa
1
.
region of each peroxidase cDNA and PSP were used in RT-PCR to study expression patterns. The
RT-PCR results Fig. 2 for GmEPa
1
were consis- tent with the above Northern blotting analysis for
GmEPa
1
, and the RT-PCR products were also confirmed by probing the products with GmEPa
1
Fig. 2. Based on the results of cDNA-specific primers, transcripts from GmEpa
2
were also de- tected in root and developing seed, and transcripts
from GmEpb
1
and GmEpb
2
were detected in root, stem, leaf, and seedpod Fig. 4.
3
.
4
. In 6itro folding of bacterially expressed GmEPa
1
proteins In order to obtain expression of soybean perox-
idases, the ORFs of GmEPa
1
and GmEPb
1
in- cluding the leader sequences were cloned into the
expression vector pET-34b + . Since the two se- quences showed the same features in both expres-
sion and in vitro folding, only GmEPa
1
is reported here. Peroxidase a
1
was found toxic to E. coli growth upon early IPTG induction, even though
the peroxidase was produced as a CBD-peroxidase fusion protein Figs. 5 and 6. The E. coli culture
containing the vector construct was then induced by IPTG at OD
600
= 1.0 to maximize the fusion
protein production. As shown in Fig. 6, a fusion protein of 60 kDa was produced and the fusion
protein was accumulated in inclusion bodies under all conditions tested. After four rounds of sonica-
tion and washing, the urea-solublized inclusion body prep gave \ 90 pure fusion proteins as
judged by SDS – PAGE Fig. 6. As can be seen in Figs. 7 and 8, the recovery of peroxidase activity
was critically dependent on the addition of hemin and on the concentration of urea, with 2 M
being optimal at pH 8.0. The addition of oxidized glutathione GSSG inhibited correct folding Fig.
8. No difference in folding efficiency existed among solubilized inclusion bodies obtained under
different bacteria growing conditions data not shown.
4. Discussion
