(Received for publication, September 26, 1996, and in revised form, November 19, 1996)
From the Biochemical Engineering Program, The gene encoding rice There is currently a strong interest in the development of new
eukaryotic hosts for the secretion of heterologous proteins. Yeasts are
attractive hosts for the production of foreign proteins because they
combine the advantages of prokaryotic and higher eukaryotic systems (1,
2). Saccharomyces cerevisiae has been used extensively for
the production of many heterologous proteins since host-vector systems,
genetic information, and recombinant DNA techniques for this organism
are well-established (1-3). However, several drawbacks have emerged in
the use of this yeast (1-5). For instance, S. cerevisiae is
regarded as a non-optimal host for the large scale production of
foreign proteins due to reduced biomass yield caused by aerobic alcohol
fermentation. In many cases, it has been difficult for S. cerevisiae to secrete large quantities of proteins 40 kDa or
larger. Hyperglycosylation of recombinant proteins is another concern.
Because of these problems, non-conventional yeasts such as Pichia
pastoris (6, 7), Kluyveromyces lactis (8), and
Hansenula polymorpha (9) have been explored as new hosts for
foreign gene expression (5).
Yarrowia lipolytica is a dimorphic yeast and is
heterothallic for mating (10). This yeast has been used to produce
citric acid, isopropylmalic acid, erythritol, and mannitol (11, 12, 13). Recently, Y. lipolytica has received special attention as a potential host for the production of heterologous proteins due to
its ability to secrete high levels of large proteins such as alkaline
extracellular protease (AEP)1 and RNase (2,
5, 10, 14). In fact, approximately 1 g/liter AEP can be secreted into
the medium under optimal conditions, indicating a significant secretion
capacity.
The XPR2 gene encoding AEP has been cloned by three
different groups (15-17). The DNA sequence of XPR2 and its
deduced amino acid sequence suggest that AEP is produced as a
prepro-protein (15, 16). The pre-region is a 15-amino acid signal
peptide followed by a stretch of 9 X-Ala or X-Pro
dipeptides (X is any amino acid) that are substrates for a
dipeptidyl aminopeptidase (18). The pro-region is composed of 122 amino
acids attached to the N terminus of the mature AEP (16). The
Lys156-Arg157 dipeptide, which is a substrate
for a KEX2-like endopeptidase encoded by the XPR6 gene, is
present at the junction between the pro-region and the mature AEP (19).
Since the promoter of XPR2 is strong and regulated by pH and
nitrogen source, the XPR2 promoter has been used to express
homologous and heterologous genes in Y. lipolytica
(20-22).
In this study, we investigated the secretion and processing of rice
Yeast strains and plasmids used in
this work are described in Table I. Plasmid pOS103 which carries the
rice
Strains and plasmids used in this work
-amylase in Oryza
sativa was expressed in the yeast Yarrowia
lipolytica, which is a potential host system for heterologous
protein expression. For efficient secretion, the strong and inducible
XPR2 promoter was used in the construction of four kinds of
expression vectors with the following configurations between the
XPR2 promoter and terminator: 1) XPR2
prepro-region-rice
-amylase coding sequence, 2) rice
-amylase
signal peptide-rice
-amylase coding sequence, 3) XPR2
signal peptide-rice
-amylase coding sequence, and 4)
XPR2 signal peptide-dipeptide stretch-rice
-amylase
coding sequence. Secretion of active recombinant rice
-amylase into
the culture medium was achieved only in the first two cases,
demonstrating that the XPR2 signal peptide is not
sufficient to direct the secretion of heterologous protein.
Furthermore, our study shows that the XPR2 prepro-region
causes imprecise processing (after
Pro150-Ala151 or
Val135-Leu136 instead of
Lys156-Arg157) and leads to N-terminal amino
acid sequences that differ from that of native rice
-amylase.
Secondary structure analysis proposed that the structural form in the
vicinity of the KEX2-like endopeptidase processing site in the
XPR2 pro-region might play a critical role in the
processing of heterologous proteins. These results suggest that the
XPR2 pro-region is dispensable for obtaining the precise N-terminal amino acid in heterologous protein secretion. In contrast, utilizing the rice
-amylase signal peptide was sufficient in directing secretion of recombinant protein with the expected N-terminal sequence, indicating that the signal peptide of rice
-amylase was
effectively recognized and processed by the Y. lipolytica secretory pathway.
-amylase in Y. lipolytica using its own signal peptide and the fusion protein with the XPR2 pre- (signal peptide)
or prepro-region. Rice
-amylase was chosen as a model protein for studying heterologous protein secretion since it has a moderate molecular mass (45 kDa), contains one N-linked glycosylation
site, and can be easily assayed.
Strains and Plasmids
-amylase cDNA at the XbaI site of pBluescript
(Stratagene, La Jolla, CA) was kindly provided by Dr. R. L. Rodriguez
(23, 24). The Escherichia coli strain DH5
(25) was used
for expression vector construction and plasmid DNA propagation. A
derivative strain of Y. lipolytica SMS397A (Mat A,
ade1, ura3, xpr2), was used as a host strain for the expression
vectors.
Designation
Descriptions
Source or Ref.
E.
coli
DH5
supE44
lacU169 (
80lacZ
M15)hsdR17 recA1 endA1 gyrA96
Ref. 29
Yarrowia lipolytica
SMS397A
Mat A, ade1,
ura3, xpr2
D. M. Ogrydziak, unpublished data
YLAMln
SMS397A harboring pXOM103-In
This work
YLAPln
SMS397A harboring pXOP103-In
This work
YLAXln
SMS397A harboring pXOX103-In
This work
YLASln
SMS397A harboring pXOS103-In
This work
Plasmids
pOS103
1.5-kilobase rice
-amylase cDNA at
XbaI site of pBluescript KS
R. L. Rodriguez (19, 27)
pIMR52
XPR2 gene in pUC19
D. M. Ogrydziak,
unpublished data
pIMR53
XPR2, URA3,
ars18 genes in pBR322
D. M. Ogrydziak, unpublished data
pIMR100
XPR2, URA3 gene in pBR322
D.
M. Ogrydziak, unpublished data
pXOM103-ln
XPR2
promoter::XPR2 prepro::rice
-amylase
in pIMR100
This work
pXOX103-ln
XPR2
promoter::XPR2 pre::rice
-amylase
inpIMR100
This work
pXOS103-ln
XPR2
promoter::rice
-amylase pre::rice
-amylase in
pIMR100
This work
pXOP103-ln
XPR2
promoter::XPR2 pre::dipeptide
stretch::rice
-amylase in pIMR100
This work
The LB medium used for E. coli cultures
was prepared as described in Sambrook et al. (25). Y. lipolytica cultures were maintained on YM medium (0.3%
Bacto-yeast extract, 0.3% Bacto-malt extract, 0.5% bactopeptone, 1%
dextrose, and 2% agar). Modified GPP medium (16) containing 1%
glycerol, 0.34% Difco proteose peptone, 50 mg/liter adenine, and
0.34% yeast nitrogen base without amino acids and ammonium sulfate was
used for the production of recombinant rice -amylase. For the first
selection of Ura+ transformants, minimal medium without
uracil was used. Selection medium for the transformants producing rice
-amylase was YPD (26), which was modified by adding 1% starch and 5 mM CaCl2, pH 6.8.
General recombinant DNA techniques were performed as described in Sambrook et al. (25). E. coli transformation was performed by the SE method (27). Yeast transformation was carried out by the lithium acetate method (28).
Selection of Transformants Producing RiceFor
the first selection, yeast cells transformed with expression vectors
were grown on minimal medium without uracil. The growing yeast cells
(Ura+) were transferred onto starch-containing YPD medium
for the second selection. After 2 days of incubation at 28 °C, the
plate was stained with iodine vapor. The transformants producing
recombinant rice -amylase were distinguished by the clear zone
around the colonies.
The starch-degrading activity of
recombinant rice -amylase was determined by monitoring reducing
sugars using the modified dinitrosalicylic acid method (23). Enzyme
solution (0.5 ml) was added to 0.5 ml of substrate solution (100 mM sodium acetate buffer with 5 mM
CaCl2 and 1% soluble starch, pH 5). After 10 min of
incubation at 30 °C, the reaction was terminated by adding 0.5 ml of
dinitrosalicylic acid solution to 0.5 ml of reaction solution and
boiled for 5 min. The solution was then diluted with 4 ml of distilled
water, and absorbance was monitored at 540 nm. As a standard, glucose
solution was used. One enzymatic unit corresponds to the amount of
enzyme required to produce 1 µmol of glucose from soluble starch per
min.
The
rice -amylase cDNA, a 1.5-kilobase insert of pOS103 (23, 24),
was exchanged with the XbaI-KpnI fragment of the
XPR2 gene in pIMR52 (Table I). The resulting plasmid,
pXO103, had the rice
-amylase cDNA inserted between the
XPR2 prepro-region and the XPR2 terminator. To
create the precise configurations, the modified SOE Polymerase Chain
Reaction (PCR) method (29) was used. The primers used for the PCR are
as follows: primer a, 5
-CATCCACCG
GGAACACAG-3
(where the underlined segment is NheI); primer b,
5
-CCG
TCCTTGTGCTCCGCCGT-3
(underlined segment is
EagI); primer 1, 5
-CTGAAACAGGACTTGTCGCTTGGCATTAGAAGAAGCAG-3
; primer 2, 5
-TCTAATGCCAAGCGACAAGTCCTGTTTCAGGGATTCAAC-3
; primer 3, 5
-GTTCAGCACCTGCATTGTTGGATTGGAGGATTGGATAGT-3
; primer 4, 5
-TCCTCCAATCCAACAATGCAGGTGCTGAACACCATGGTG-3
; primer 5, 5
-ACTGCCGTTCTGGCCCAAGTCCTGTTTCAGGGATTCAAC-3
; primer 6, 5
-CTGAAACAGGACTTGGGCCAGAACGGCAGTGAGAATAGT-3
; primer 7, 5
-CTGAAACAGGACTTGAGGCACAGCAGCAGGGGCAGCATC-3
; primer 8, 5
-CCTGCTGCTGTGCCTCAAGTCCTGTTTCAGGGATTCAACTGG-3
.
To construct the hybrid between the XPR2 prepro- and rice
-amylase coding sequence, the first two PCRs were performed
separately. One PCR mixture contained primers a and 1 with pIMR52 as a
template, and the other PCR mixture had primers b and 2 with pOS103 as
a template. After the first PCR products were purified, a second PCR
was carried out with primer a, primer b, and the two purified PCR
products as templates. For the other three fusions, the same PCR method
was utilized with different primers (primers 3 and 4, 5 and 6, and 7 and 8 were used for pXOS103, pXOX103, and pXOP103, respectively). The
final PCR fragments, which had NheI and EagI restriction enzyme sites at the 5
and 3
ends, respectively, were
purified and exchanged with the small fragment of pXO103 digested with
NheI and EagI. Consequently, four precise fusion plasmids were constructed (pXOM103, pXOX103, pXOP103, and pXOS103). The
PCR conditions were as follows: 1 µmol of each primer and 5 units of
Vent polymerase (New England BioLabs, Beverly, MA) were used for each
reaction in 100-µl total volume. The PCR reactions were performed for
25 cycles of 1 min at 94 °C, 2 min at 60 °C, and 3 min at
72 °C, followed by a 7-min incubation at 72 °C.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described by Laemmli (30). Samples were resolved on 10% polyacrylamide gels containing 0.1% SDS at room temperature. After electrophoresis, gels were stained with Coomassie Brilliant Blue R-250 and dried on cellulose acetate film. Amylolytic activity was detected by repeatedly rinsing the gels with distilled water for 15 min at room temperature to remove SDS. The gels were then immersed in the enzyme reaction buffer (50 mM sodium acetate, 5 mM CaCl2, pH 5) containing 1% (w/v) soluble starch for 30 min at room temperature. The gels were rinsed with the same buffer without soluble starch for 15 min, stained with iodine solution (0.3% iodine, 3% potassium iodide) for 10 min, and rinsed with distilled water. Finally, amylolytic activity was detected as a clear zone in a brown background. The N-terminal amino acid sequence was determined by an automated Edman degradation apparatus (model 477A, Applied Biosystems) with on-line high performance liquid chromatography (model ABI 120). One µg of each sample was separated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane. After staining with Coomassie Brilliant Blue R-250, the sample bands were cut and used for sequencing.
Prediction of the Protein Secondary StructurePrediction of AEP and hybrid protein secondary structures were performed by the profile network method called PHDsec (Protein Design Group, European Molecular Biology Laboratory, Heidelberg, Germany). The secondary structure prediction method is rated at 72.1% average accuracy for water-soluble globular proteins in the three states, helix, strand, and loop.
Western Blot AnalysisAfter separation by SDS-PAGE,
proteins were electroblotted onto a nitrocellulose membrane (Schleicher
& Schuell) in ice-cold transferring buffer (15.6 mM Tris,
120 mM glycine, and 20% methanol, pH 8.3) at 100 V for
1 h. The first antibody for Western blot analysis was anti-barley
-amylase antibody which was generously given by Dr. S. Katoh and Dr.
M. Terashima (Kyoto University, Japan). The second antibody was
anti-rabbit IgG antibody conjugated with alkaline phosphatase supplied
by Vector (Burlingame, CA). The detection procedure followed was the
same as that recommended by the manufacturer.
Recombinant rice -amylase
produced by the Y. lipolytica transformants was purified
using
-cyclodextrin-substituted, epoxy-activated Sepharose 6B
affinity chromatography (23, 31). The recombinant strain was grown in
GPP medium with a working volume of 1 liter at 28 °C for 30 h
in a 2-liter jar fermentor. After centrifugation (5000 rpm with Sorvall
GSA rotor, 10 min at 4 °C), the secreted proteins in the supernatant
were precipitated with 75% (w/v) ammonium sulfate. A protein pellet
was obtained by centrifugation (5000 rpm with Sorvall GSA rotor, 15 min
at 4 °C) and redissolved into the buffer (50 mM sodium
acetate, 5 mM CaCl2, pH 5). After dialysis, 40 ml of protein solution was subjected to
-cyclodextrin affinity chromatography. After the sample was applied to a column (2.0 × 10.0 cm), the column was washed with 50 ml of low salt buffer (50 mM sodium acetate, 5 mM CaCl2, 5 mM NaCl, pH 5) and 50 ml of high salt buffer (50 mM sodium acetate, 5 mM CaCl2, 0.3 M NaCl, pH 5). The rice
-amylase was eluted with 8 mg/ml
-cyclodextrin, and each fraction volume was 5 ml.
To direct rice -amylase through the Y. lipolytica secretory pathway, four kinds of expression vectors,
pXOM103, pXOX103, pXOP103, and pXOS103, were constructed (see
"Materials and Methods") in which the strong and inducible
XPR2 promoter was used with the following configurations
between the XPR2 promoter and terminator: 1) XPR2
prepro-region-rice
-amylase coding sequence, 2) rice
-amylase
signal peptide-rice
-amylase coding sequence, 3) XPR2 signal peptide-rice
-amylase coding sequence, and 4) XPR2
signal peptide-dipeptide stretch-rice
-amylase coding sequence. To
create the final integrative vectors for Y. lipolytica,
fusion gene fragments from pXOM103, pXOX103, pXOP103, and pXOS103 were
inserted into pIMR100 (Table I) to construct pXOM103-In,
pXOX103-In, pXOP103-In, and pXOS103-In (Fig. 1).
Plasmids pXOM103-In, pXOX103-In, pXOP103-In, and pXOS103-In were
independently transformed into Y. lipolytica SMS397A.
Chromosome integration of the plasmids into the genome of the host
Y. lipolytica strains was confirmed by Southern blot
analysis using the rice -amylase cDNA as a probe (data not
shown). The resulting transformants were named YLAMIn (
ipolytica producing rice
-
mylase with
pXO
103-
), YLAXIn, YLAPIn, and YLASIn,
respectively. The secretion of recombinant rice
-amylase was
determined with the plate clear zone assay described under "Materials
and Methods." After 2 days of incubation at 28 °C, transformants
producing rice
-amylase were detected by the clear zone around the
colonies (Fig. 2). YLAXIn and YLAPIn were not able to
produce rice
-amylase either intracellularly or extracellularly
(Fig. 2), implying that the signal peptide cleavage sites in the fusion
proteins of pXOX103 and pXOP103 were not recognized efficiently. In
contrast, YLAMIn and YLASIn showed clear zones (Fig. 2) and
-amylase
activity in the culture medium, indicating secretion of recombinant
rice
-amylase. However, the plate clear zone and rice
-amylase
assays demonstrated that YLASIn strains produce higher amounts
(~3-fold) of rice
-amylase than YLAMIn strains. Since YLAMIn and
YLASIn produced recombinant rice
-amylase, the secreted proteins
from both strains were studied further.
The Rice
The presence of
recombinant rice -amylase in the YLAMIn culture supernatant was
confirmed by SDS-PAGE (Fig. 3) and Western blot analysis
(Fig. 4). The rice
-amylase secreted by YLASIn was
purified by
-cyclodextrin affinity chromatography (Table II). Figs. 3 and 4 show that the purified recombinant
rice
-amylase is homogeneous with a molecular mass of 45 kDa, which
is similar to the native enzyme produced in germinating rice seed (23). However, the specific activity of purified rice
-amylase was 143 units/mg, which is lower than reported for S. cerevisiae
(226 units/mg) (23). The difference in specific activities may be due
to variations in the methods used for determining total protein concentration and enzyme activity. To determine glycosylation and the
size of the N-linked carbohydrate chain, purified rice
-amylase was treated with endoglycosidase H, which cleaved the N-linked oligosaccharide chain from the glycoprotein. Fig.
3A demonstrates that the N-linked glycosyl group
is approximately 3 kDa and that deglycosylation does not affect enzyme
activity (Fig. 3B).
|
To determine the signal peptide cleavage site of recombinant rice
-amylase, N-terminal amino acid sequence analysis was performed. Previous attempts to determine the N-terminal amino acid sequences of
native and recombinant (S. cerevisiae) rice
-amylases
have failed (23). However, a tentative signal peptide processing site
between Gly31 and Gln32 has been proposed based
on a comparison with the amino acid sequence of barley
-amylase (23,
24). The recombinant rice
-amylase produced by YLASIn had
Gln-Val-Leu-Phe-Gln as an N-terminal amino acid sequence, which is
identical to the proposed sequence of native rice
-amylase (Fig. 6).
Although the XPR2 signal peptide with and without a
diaminopeptide stretch failed to direct the secretion of rice
-amylase (Fig. 2), our results indicate that the signal peptide of
rice
-amylase is recognized and efficiently processed in the
secretory pathway of Y. lipolytica. In fact, the rice
-amylase signal peptide is the first exogenous signal sequence
reported that conducts heterologous protein secretion in Y. lipolytica.
The XPR2 Prepro-region Causes Imprecise N-terminal Sequences in Recombinant Rice
When the rice -amylase produced
by YLAMIn was purified by affinity chromatography, two distinct bands
appeared on the SDS-PAGE gel (Fig. 5A).
Interestingly, both bands showed enzyme activity (data not shown)
and reacted with the anti-barley
-amylase antibody (Fig.
5B). Molecular sizes of major and minor recombinant rice
-amylases were approximately 45 and 47 kDa, respectively. After endoglycosidase H treatment, two bands still appeared, and the size
difference between the two bands remained unchanged, indicating that
glycosylation does not confer any difference between the two proteins.
Moreover, the specific activity of those proteins (136 units/mg) was
almost the same as that of the recombinant rice
-amylase produced by
YLASIn.
To investigate the processing of recombinant rice -amylase secreted
by YLAMIn, the N-terminal amino acid sequences of the two proteins
appearing in SDS-PAGE were determined. Both proteins had unexpected
N-terminal amino acid sequences. Initially, we predicted that the
recombinant rice
-amylase produced by YLAMIn might be processed
after Lys156-Arg157 at the end of the
XPR2 pro-region, which is a substrate for a KEX2-like
endopeptidase encoded by the XPR6 gene (19). However, results show that the major and minor proteins are processed after Pro150-Ala151 and
Val135-Leu136, respectively (Fig.
6).
To determine whether or not the secondary structure of the XPR6
processing site (Lys156-Arg157) in AEP was
altered in the hybrid protein, computer analysis of the protein
secondary structure was carried out. In the predicted secondary
structure of AEP, the Lys156-Arg157 region is a
loop exposed to the surface (data not shown). Interestingly, however,
the structure of the same region in the hybrid protein, in which the
XPR2 prepro-region was fused with the rice -amylase coding region, is an
-helix buried from the surface. These data imply that the XPR6 cannot access the
Lys156-Arg157 cleavage sites in the hybrid
protein. Therefore, we have concluded that the structural change in the
XPR6 cleavage site between AEP and the hybrid protein directs an
altered processing of recombinant rice
-amylase. However, there is
no known protease that reacts with the cleavage site described above.
Hence, the enzyme that may be involved in the processing of recombinant
rice
-amylase secreted by YLAMIn remains an open question. From
these results, we have concluded that even if the prepro-region of
XPR2 were successfully employed to direct the secretion of
some heterologous proteins in Y. lipolytica (20), it may
result in imprecise cleavage of the prepro-fusion protein and produce
the wrong N-terminal amino acid in the secreted protein; thus, the
XPR2 prepro-region is dispensable for foreign protein
secretion in Y. lipolytica.
Since Yarrowia lipolytica is an attractive host for the
production and secretion of heterologous proteins (5, 14), it has been
used to secrete foreign proteins such as bovine prochymosin, S. cerevisiae invertase, porcine -interferon, and human blood coagulation factor XIIIa (20-22, 32). In all cases, the
XPR2 signal sequence was used for directing endoplasmic
reticulum translocation. However, utilizing the XPR2 signal
peptide for heterologous protein secretion is not always successful as
demonstrated in this study. Furthermore, we found that although the
prepro-region of XPR2 was sufficient to direct secretion of
recombinant rice
-amylase, the signal peptide (pre-region) of
XPR2 by itself and with a dipeptide stretch did not direct
secretion of rice
-amylase. Instead, the heterologous signal peptide
of rice
-amylase was efficiently recognized and processed by the
Y. lipolytica endoplasmic reticulum translocation machinery,
and it conducted foreign protein secretion in this yeast. This result
demonstrates that the signal peptide of rice
-amylase can be used
for achieving heterologous protein secretion in Y. lipolytica.
The results of this study show that the recombinant -amylase
produced by YLASIn is a 45-kDa glycoprotein, which is similar to native
rice
-amylase, with Gln-Val-Leu-Phe-Gln as an N-terminal amino acid
sequence. Previously, the N-terminal amino acid sequence of native and
recombinant rice
-amylases could not be determined because the
N-terminal amino acid was modified or blocked (23). However, the site
between Gly31 and Gln32 was proposed as a
signal peptide cleavage site based on the sequence comparison to barley
-amylase (23, 24). Our data confirm that this proposed signal
peptide cleavage site is correct.
Interestingly, two types of active recombinant rice -amylase were
secreted by YLAMIn. Using endo H treatment and N-terminal amino acid
sequence analysis, it was shown that the two secreted recombinant
proteins are processed differently (Figs. 5 and 6). Tokunaga et
al. (33) also detected a minor portion of mouse
-amylase which
was processed differently when it was expressed and secreted by
S. cerevisiae using the pGKL128-kDa killer toxin secretion
signal sequence. They assumed that the secreted
-amylase was
processed by a second protease in the secretory pathway after removal
of the signal sequence. Since our results show that the XPR6 cleavage
site (Lys156-Arg157) is not processed in the
XPR2 prepro-rice
-amylase fusion protein, it was presumed
that an alternative protease might attack the fusion protein instead of
XPR6. However, we failed to identify a specific protease that may be
involved in this processing. Interestingly, Valverde et al.
(34) found a putative
-helix structural motif at the C-terminal of
the pro-region which might play a critical role in directing the
secretion of active endothiapepsin in S. cerevisiae. In this
study, we utilized secondary structure computer analysis for fusion
proteins to determine if this is the case. Although secondary structure
computer analysis revealed that the
-helical structure is not
conserved in the C terminus of the AEP pro-region, the predicted
secondary structure of the hybrid protein shows that the
Lys156-Arg157 site is altered from the exposed
loop form in AEP to the buried helical structure in the fusion protein.
Therefore, we presume that the XPR6 cannot access the
Lys156-Arg157 cleavage sites in the hybrid
protein. In conclusion, although the pro-region is known to play an
essential chaperon-like role in the secretion of several proteinases
and is indispensable for the secretion of AEP (35-37), the AEP
pro-region is unnecessary for the secretion of heterologous protein in
Y. lipolytica because it can cause imprecise processing of
the hybrid protein and lead to the wrong N-terminal amino acid in the
final product.
We thank Dr. R. L. Rodriguez (University of
California, Davis) for providing us with the plasmid pOS103, Dr. S. Katoh (Kyoto University, Japan) for providing us with anti-barley
-amylase antibody, and Dr. M. Terashima (Kyoto University, Japan)
for his helpful suggestions. We also thank Dr. Y. M. Lee (Protein
Structure Laboratory at the University of California, Davis) for
N-terminal amino acid sequencing, Sam Matoba, and I. H. Lee (University
of California, Davis) for very helpful discussions, and Caroline Crispino for her assistance in manuscript preparation.