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Journal of General Microbiology (1987), 133, 1775-1782.
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1775
Secretion of Bacillus subtilis a-Amylase in the Periplasmic Space of
Escherichia coli
By K O H - I C H I T A C H I B A N A , ' K O J I Y O D A , ' S A T O R I W A T A N A B E , '
HIROSHI KADOKURA,' YOSHIHIRO KATAYAMA,' K U N I O YAMANE,*
MAKARI YAMASAKI'* AND G A K U Z O TAMURA'
Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo,
Bunkyo-ku, Tokyo 113, Japan
Institute of Biological Sciences, University of Tsukuba, Sakura, Ibaraki-ken 305, Japan
(Received 27 October 1986 ;revised 23 February 1987)
The Bacillus subtilis a-amylase structural gene (amyE) lacking its own signal peptide coding
sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA)signal peptide
coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On
induction of the phoA promoter, the B. subtilis a-amylase was expressed and almost all the
activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal
amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein
was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.
INTRODUCTION
When useful proteins are produced in Escherichia coli, it is preferable that they are secreted
into the periplasmic space or the culture medium because (1) their detection, recovery and
purification will be easier, (2) the amount of the product may increase, and (3) degradation of the
product by cytoplasmic proteases will be avoided. Secretory or membrane proteins are generally
synthesized as large precursors with a signal peptide at the amino-terminus. The early steps of
secretion are initiated by the function of the signal peptide. During translocation across the
plasma membrane, the signal peptide is proteolytically cleaved to yield the mature protein
(Blobel & Dobberstein, 1975; Davis & Tai, 1980; Michaelis & Beckwith, 1982). Hence, in the
secretory production of proteins, it is necessary to endow them with a signal peptide. A series of
secretion vectors for the expression and secretion of foreign gene products in E. coli has been
constructed and are named 'Psi vectors', because they contain the promoter and the signal
sequence of the E. coli alkaline phosphatase gene (phoA).They also have unique cloning sites just
at the end of the signal sequence. The human a-interferon gene (Miyake et al., 1985) and the
human epidermal growth factor gene (Oka et al., 1985) have been inserted just behind thephoA
signal sequence. In both cases the protein is found in the periplasmic space. B. subtilis a-amylase
is a well-characterized enzyme that has industrial uses. The structural gene (amyE), including
the promoter and the signal sequence, has been cloned in PUB1 10 (Takeichi et al., 1983)and the
sequence determined (Ohmura et al., 1983). It is of interest to elucidate whether the mature
polypeptide of the B. subtilis a-amylase can be translocated to the periplasm with the aid of the E.
coli signal peptide, and whether the E. coli signal peptidase can correctly process the fusion
protein or not. In this paper, the coding sequence of the a-amylase has been subcloned into the
'Psi vector', and expression and secretion of the protein into the periplasm has been examined.
METHODS
Bacterialstrains,media andplasmids. E. coli K12 MC1061 [F-araD139 A(ara-le~)~~~~lacX74(AlacIPOZY)
galU
galK hsdM+hsdR- rpsL] was used as the general host for transformation, expression and experiments. E. coli
K12 LE392 [F-supE supF hsdM+ hsdR- lac+]was also used as the host for expression and secretion experiments.
0001-3780 0 1987 SGM
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K.-I. TACHIBANA A N D OTHERS
E. coli K12 YK660 [F196+thi-1 leuB6 metE70 hisF860 lacZ36 xyl-5 rpsLlO5 rpsElSphoA8 recAl] was used for
production of the single-stranded plasmid DNA derivatives. Transformation and plasmid construction were done
as described by Maniatis et al. (1982). Nutrient broth containing 1.0% (w/v) peptone, 0.3% (w/v) meat extract and
0.5% (w/v) NaCl was purchased from Eiken and used as rich medium generally. The inducing medium forphoAdirected production of the gene product was the MOPS medium of Neidhardt et al. (1974) supplemented with
about 0.4 mM-sodium phosphate as an endogenous component of 0.2% (w/v) Difco vitamin-free Casamino acids.
For induction of b-galactosidase the medium was also supplemented with 1 m-isopropyl b-thiogalactoside
(IPTG) and 5 mM cyclic AMP. The cells consume sodium phosphate in the medium as they grow. When the ODSso
reaches about 0.6, the concentration of sodium phosphate becomes low enough to derepress the phoA promoter.
The repressing medium was the same as above except that 1 mwsodium phosphate was added.
The a-amylase structural gene (amyE) of B. subtilis Marburg NA64 has been cloned into PUB1 10 (Takeichi et
al., 1983). This plasmid was designated pTUB4 and the DNA sequence of the inserted 2.3 kb fragment is known
(Ohmura et al., 1983). pYK33 I(a 'Psi vector') is a derivative of pBR322 containing the same multiple cloning sites
as M13mplO (Norrander et al., 1983) at the end of the promoter-signal sequence and the intergenic region of the
filamentous fl phage (Beck & Zink, 1981). The construction of pYK331 will be reported elsewhere. The singlestranded circular DNAs were prepared from filamentous-phage-like particles which were produced after the
infection of male E. coli harbouring pYK331 or its derivatives with fd phage.
Enzymes and chemicals. All enzymes were purchased from Takara Shuzo (Kyoto, Japan). Reaction conditions
used were those described by the manufacturer. IPTG, cyclic AMP, MOPS and Tricine were purchased from
Sigma. Vitamin-free Casamino acids were from Difco. [3SS]Methionine[400 Ci mmol-l (14.8 TBq mmol-l)] was
purchased from Amersham. The B. subtilis a-amylase antibodies were a kind gift from Dr Y.Nagata (Institute of
Applied Microbiology, University of Tokyo).
Preparation of theperiplasmic fraction. This was done by the osmotic shock method of Neu & Heppel (1965).
SDS-PAGE. The buffer system was that of Laemmli (1970). Proteins were detected by staining with Coomassie
Brilliant Blue or by autoradiography.
Immunological detection of a-amylase. The labelling of cells with [3sS]methionineand the detection of a-amylase
by immunoprecipitation was done as described by Ito et al. (1981).
Synthesis of oligonucleotide and site-specijc deletion. A 2 1-mer oligonucleotide (S'TTCGCCGTTTCAGCTTTTGTC3') was prepared by a manual solid-phase method (Miyoshi et al., 1980). The site-specificdeletion was done
as described by Wallace et al. (1980) except that single-stranded DNA was prepared from single-stranded phage
particles.
Assay of enzymes. a-Amylase was assayed by the method of Fuwa (1954). Alkaline phosphatase was assayed by
the method of Brickman & Beckwith (1975). b-Galactosidase was assayed by the method of Miller (1972).
Purification and amino acid sequence analysis of the secreted a-amylase. The a-amylase secreted into the
periplasmic space was purified as described by Matsuzaki et al. (1974). Amino acid sequence analysis was done by
Edman degradation using a solid-phase automated sequencer (Beckman system 890). The phenylthiohydantoinamino acid derivatives were analysed by HPLC (Beckman Model 1 IOA).
RESULTS
Cloning of the amyE gene in the E. coli expression vector
In the course of studies on secretion of foreign products in E. coli, expression of the amyEgene
with the aid of the promoter and signal sequence of the phoA gene was attempted. First pTUB4
was cleaved with BglII and XbaI, and the 4.2 kb fragment containing the amyE gene was
inserted between the BumHI and XbaI sites in the multiple cloning sites of pYK331. E. coli
K 12 MC 1061 was transformed by the ligation mixture and ampicillin-resistant transformants
were selected. They were then transferred to a nutrient broth agar plate containing 1% (w/v)
soluble starch and, after growth at 37 "C overnight, the plate was exposed to iodine vapour.
Colonies carrying the new plasmid, designated pYK338, were detected by the formation of large
haloes. E. coli K12 MC1061 carrying pYK331 or pYK338 was grown in a liquid culture under
inducing or repressing conditions and the cells and supernatant were separated by
centrifugation. Cells were subjected to osmotic shock to obtain the periplasmic fraction.
a-Amylase activity of MC1061/pYK338 was 2.4 units ml-I (ODssounit)-' and increased to
9.2 units ml-I (ODsso unit)-' during induction. Of the total activity 54% was detected in the
periplasm and little in the medium. From the sequence analysis of DNA (Ohmura et al., 1983;
Yang et al., 1983) and protein (Mantsala & Zalkin, 1979), the product of umyE in B. subtilis was
predicted to have an amino-terminal extension of 41 amino acid residues not found in the
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Secretion oja-amylase in E. coli
Xbal
Xhl
(b)
+ BamHI
Bglll
+ Xbal
phoA signal (21a.a) ------Intervening
.-b
sequence-
+.let Lys C l n Ser . . . . . . . . . . Thr Lys Ala T r p Ala Ala G l y Arg k u Lhnb
C T C AAA CAA ACC . . . . . . . . . . ACA T-A
TCC C C T CCA C C T CCA C T C TAG
Hind111
-About
1 kb
-
om) E
I
...
...
signal (33a a) ___
. . . . . . . . . . . . . . . . . . . . . . . . . . MCt Phe Ala Lys . . . . . . . . . . . Pro Ala Ala Ala
...............
33 A 34
A C T C T C A A C A A T C T T T CCA AAA
...........
C F C C C C C T CCC
Hpal I
unt.,’€
41
42
Ser A 1 a ) G l u Thr Ala Asn Lys Ser Asn G 1 u ’ L . e ~ Thr Ala
A C T C C T CAA ACC CCC AAC AAA TCG A A T GAG C T T ACA C C A
AIul
..................
..................
Fig. 1 . Construction and structure of pYK338. (o)phoA’ represents the promoter and the coding regions
for the signal peptide of the E. colialkaline phosphatase gene; amyErepresents the structural gene of the
B. subtilis a-amylase; sfp represents the intergenic region of the filamentous fl phage (see Methods);
MCS represents the multiple cloning sites. (b)Translational reading frame of the phoA’-umyE chimera
gene in pYK338. The amino-terminus of the extracellular mature a-amylase is indicated by the arrow
labelled B; the predicted cleavage site of the signal peptidase is indicated by the arrow labelled A.
secreted enzyme. This sequence contains a signal sequence for secretion and is first cleaved
between Ala and Glu (33rd and 34th amino acid from the amino-terminus, position A in Fig. 1 b)
during the secretion process and further cleaved between Glu and Leu (position B in Fig. 1b) to
form the mature extracellular a-amylase. If position A of the amyE gene is joined to just after the
end of the phoA signal codons to form an ‘in frame’ gene fusion, a-amylase should be expressed
and secreted under the control of the promoter and signal sequence of thephoA gene. A deletion
between the end of the phoA signal sequence and position A of the amyE gene in pYK338 was
therefore sought by a site-specific deletion using a synthetic oligonucleotide (Fig. 2).
Restriction endonuclease trimming oj’ the intervening sequence
Several attempts to delete about 1000 bases from pYK338 directly with the aid of a mutagenic
21-mer oligonucleotide failed. This might be explained by a marked lowering of the probability
of annealing between the primer and the template, so, to facilitate the deletion, a major part of
the intervening DNA was deleted with the aid of restriction enzymes (Fig. 2). pYK338 was cut
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K.-I. T A C H I B A N A A N D O T H E R S
H
p
u
n
SUII
l
?
EcoRl (partially)
T,-ligase
I
17h
( Hbtd I I I iHnal I 1
Helper
phage fd
stranded
ApY K 3.78
Fig. 2. Construction of pAMYA. phoA’ represents the promoter and the coding regions for the signal
peptide of the E. coli alkaline phosphatase gene; amyE represents the structural gene of the B. subtilk aamylase; sfp represents the intergenic region of the filamentous f t phage; MCS represents the multiple
cloning sites. pYK338 was converted to ApYK338 by restriction enzyme trimming and it was further
converted to pAMYA by oligonucleotide-directed deletion. The latter deletion was facilitated by
making ApYK338 into a single-stranded circular form by fd phage function.
with Sun and a 1900 bp fragment containing the amino-terminus of the amyEgene was purified.
This fragment was cut with HpaII and the staggered ends were filled in using DNA polymerase
Klenow fragment. After cutting with EcoRI, the 450 bp fragment was purified; it has one blunt
and one EcoRI-compatible end. Separately, pYK338 was cut with HindIII, the staggered ends
filled in using DNA polymerase Klenow fragment, and, after cutting partially with EcoRI,
ligated to the 450 bp fragment. This new plasmid (ApYK338) is about 1000 bp shorter than
pYK338. The strain carrying ApYK338 did not produce a-amylase because of the reading frame
shift for the amyE gene.
Oligonucleotide-directed site-specific deletion
The single-stranded derivative of ApYK338 was prepared as described in Methods. The
synthetic 21-mer oligonucleotide was 5’-phosphorylated and used as a primer for DNA synthesis
on the single-stranded circular DNA. E. coZi MC1061 was transformed with the reaction mixture
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Secretion of a-amylase in E. coli
Table 1. Distribution of a-amylase and marker enzymes in E. coli K12 LE392 (lac+)
All three enzymes were assayed in the same culture. The results are mean values f SD of five
independent experiments.
Enzyme activity*
Enzyme
Plasmid
a- Amylase
None
pYK331
pYK338
pAMYA
Alkaline
phosphatase
None
pYK331
pYK338
pAMYA
B-Galactosidase
None
pYK331
pYK338
pAMYA
Total
Periplasmic
0.1
0
0.1
0.1
1.1 f 0.1
8.6 f 1.0
0.7 f 0.1
56.5 f 3.0
0.1
10.1 f 0.7
0.2
9.7 f 0.5
0.1
11.4 f 1.1
0
0.1
0
0
1.0 f 0.1
7.8 f 0.5
0.7 f 0.1
55.4 f 2.8
0
9.8 f 0.5
0.2
9.3 f 0.5
0.1
10.9 f 1.0
0
10.4 f 0.9
0.1
0-2
0.1
10.9 f 1.0
33.3 f 1.5
47.3 f 3.6
42-9 f 2.6
58.8 f 3-8
43.5 f 2.2
49.3 f 3.1
45.4 f 2.6
61.4 f 2-9
0.1
0
0.1
0.7 f 0-1
0.1
0.2
* a-Amylase activity, units ml-l (ODSSounit)-';
x pgalactosidase activity, units ml-l (ODSS0)-l;
alkaline phosphatase activity, AAd2,-, min-' ml-I (ODSso unit)-'.
and transformants were selected for ampicillin resistance. Some transformants should carry
mutant plasmids. Transformants were mixed, plasmid DNAs prepared from them, and digested
to completion with Hind111 which should not cut the deletion plasmid. This mixture was used to
transform E. coli YK660; ampicillin-resistant transformants were selected and tested for aamylase on inducing agar plates containing 1% (w/v) starch. Of fifty transformants tested three
gave large haloes on exposure to iodine vapour.
Characterization of the mutant plasmid
The three transformants were purified and plasmid DNA prepared from them. Singlestranded derivatives of the plasmids were prepared by superinfection with fd phage. The DNA
sequence covering the junction of the phoA and the amyE genes was determined by the dideoxy
chain-termination method (Sanger et al., 1977) and was found to be identical for all three
plasmids. The direct and 'in frame' linkage of the two genes was also verified. One of these
plasmids (pAMYA) was examined further.
Enzyme localization
a-Amylase and other marker enzyme activities were assayed in each culture after subcellular
fractionation : alkaline phosphatase, a typical periplasmic enzyme, was found entirely in the
periplasmic fraction (Table 1) and /?-galactosidase,a cytoplasmic enzyme, was absent both from
periplasm and medium. As E. coli K12 MC1061 is a lacX74 deletion mutant, E. coli K12 LE392
(lac+)transformed with pYK331, pYK338 or pAMYA was used in this experiment. In the
inducing medium, the strain carrying pAMYA produced 7-fold higher activity than those
carrying pYK338. Almost all the activity was found in the periplasmic fraction and not in the
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K.-I. TACHIBANA A N D OTHERS
Fig. 3. Autoradiogram of 12.5% (w/v) SDS-PAGE of [3SS]methionine-labelledwhole cells or the
periplasm fraction from strains LE392/pYK33 1 or LE392/pAMYA. Cells were labelled with
[3sS]methioninefor 2 h, harvested, and used for enzyme assay and product analysis by SDS-PAGE.
Lanes 1, 2, 5, 6, 9 and 10, pYK331; 3, 4, 7, 8, 11 and 12, pAMYA; 1-4, whole cells; 5-8, periplasm
fraction obtained by osmotic shock ; 9-1 2, anti-a-amylase antiserum immunoprecipitate of periplasm
fractions; 2,4,6,8,10 and 12, inducing conditions; 1,3,5,7,9 and 11, repressing conditions. The bands
of a-amylase are indicated by the arrow.
medium after subcellular fractionation. The a-amylase activity was entirely in the periplasm.
These results show that the promoter and the signal sequence ofphoA in pAMYA can efficiently
direct the production and secretion of the B. subtilis a-amylase into the periplasm of E. coli. To
examine the products from pAMYA, cells carrying pAMYA or pYK331 were labelled with
[3SS]methionineand subjected to SDS-PAGE after subcellular fractionation. Each periplasmic
fraction was immunoprecipitated with rabbit anti-a-amylase antiserum and was analysed by
SDS-PAGE (Fig. 3). In lanes of the whole cell lysate and the periplasmic fraction of pAMYA
under inducing conditions, aspecific band with an M , value corresponding to the B. subtilis aamylase (59 x lo3) was found on the autoradiogram (Fig. 3, lanes 4 and 8). In the
autoradiogram of immunoprecipitates, a specific band was detected only in the cells carrying
pAMYA under inducing conditions (lane 12). The a-amylase in the periplasmic fraction was
purified and the five amino-terminal residues shown to be Glu-Thr-Ala-Asn-Lys-. This
sequence is that predicted if the precursor polypeptide is correctly processed by the E. coli signal
peptidase.
DISCUSSION
As shown in Table 1, a small amount of B. subtilis a-amylase was synthesized and secreted into
the periplasm of E. coli carrying pYK338 even under the repressed condition of the phoA
promoter. It is quite possible
that the promoter of the B. subtilisamyEgene can function in E . coli
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Secretion of a-amylase in E. coli
1781
because the sequence of the promoter does not differ from the consensus sequence of the E. coli
promoter (Hawley & McClure, 1982). The B. amyloliquefaciensa-amylase gene is also expressed
in E. coli (Palva et al., 1981). The increased a-amylase activity in strain LE392/pYK338 in
derepressing conditions may be due to considerable readthrough from the phoA promoter.
The trace of a-amylase activity recorded for strains which should not possess the amyE gene is
probably due to some reducing substances which influence colour development in the assay.
This is a disadvantage of this assay method. It is a curious phenomenon that b-galactosidase
activities increased slightly in low-phosphate media (Table 1). The promoter of the lac operon
may be further activated or some upstream promoter may be derepressed in this strain.
A single large protein (M,54 x lo3) is present in the periplasmic extract in repressing
conditions (Fig. 3). It should be different from elongation factor Tu ( M , 44 x lo3), which is
released by osmotic shock and represents a major protein in some cases (Jacobson &
Rosenbusch, 1976), but its nature is as yet unknown. The extra bands at an M , of about 24 x lo3
in lane 6 should be products of some open reading frame downstream of thephoA promoter in
pYK33 1, because their expression is enhanced under derepressed conditions and eliminated
when the amyE gene is inserted behind the phoA promoter.
Our results show that B. subtilis a-amylase was efficiently expressed and secreted into the
periplasm of E. coli with the aid of the phoA promoter and signal sequence. Production of aamylase on inducing agar plates containing starch could be easily detected by halo formation on
exposure to iodine vapour. This halo formation was probably due to some cell lysis, because aamylase was not detected in the liquid culture medium (Table 1). The DNA sequence of the B.
subtilis a-amylase is known so the amino acid sequence of its precursor protein can be deduced
(Ohmura et al., 1983). It has an amino-terminal extension of 41 amino acid residues. If this
sequence is the signal peptide, it is much longer than signal peptides found in other exported
proteins of prokaryotes and eukaryotes (Kreil, 1981). Possibly only a part of this sequence forms
a signal peptide. For this reason it has been suggested that the precursor protein of the B. subtilis
a-amylase may be cleaved twice to form the mature extracellular enzyme (Ohmura et al., 1983;
Yamazaki et al., 1983), as reported for the formation of exopenicillinase of Bacillus licheniformis
(Nielsen & Lampen, 1982) and Staphylococcus aureus (McLaughlin et al., 1981). In the present
study we constructed a plasmid, pAMYA, in which the end of the phoA signal sequence is
directly linked to the predicted first cleavage site of the B. subtilis a-amylase sequence, using the
technique of oligonucleotide-directed site-specific deletion, and examined whether the
precursor protein synthesized by thephoA’-amy’E gene is cleaved twice or not in E. coli like the
extracellular a-amylase in B. subtilis. From the sequencing analysis of the five amino-terminal
residuk of the secreted a-amylase in the periplasm, it turned out that the product was correctly
processed by the E. coli signal peptidase at the end of the phoA signal sequence and did not
undergo the second processing. The double processing in secretion may be peculiar to bacilli and
staphylococci.
We are grateful to Y. Nagata for his generous gift of the rabbit anti-a-amylase antiserum. This work was
supported in part by a Grant-in-Aidfor Scientific Research from the Ministry of Education, Science and Culture
of Japan and in part by a research grant from the Institute of Physical and Chemical Research, Japan.
REFERENCES
DAVIS,B. D. & TAI,P. C. (1980). The mechanism of
BLOBEL,G.& DOBBERSTEIN,
B. (1975). Transfer of
protein secretion across membranes. Nature, London
proteins across membranes. I. Presence of proteo283,433-438.
lytically processed and unprocessed immunoglobulin
light chains. Journal of Cell Biology 67, 835FUWA,H. (1954). A new method for microdetermination of amylase activity by the use of amylose as the
851.
substrate. Journal of Biochemistry 41, 583-603.
BECK,E. & ZINK,B. (1981). Nucleotide sequence and
genome organisation of filamentous bacteriophage
€ AWLEY, D. K . & MCCLURE,
W. R. (1983). Compilation and analysis of Escherichia coli promoter DNA
fl and fd. Gene 16, 35-58.
sequences. Nucleic Acids Research 11, 2237-2255.
BRICKMAN,E. & BECKWITH,J. (1975). Analysis of the
regulation of Escherichia coli alkaline phosphatase I 0, K., BASSFORD,P. J. & BECKWITH,J. (1981).
Protein localization in E. coli: is there a common step
synthesis using deletions and 480 transducing
phages. Journal of Molecular Biology 96, 307in the secretion of periplasmic and outer-membrane
316.
proteins? Cell 24, 707-717.
Downloaded from www.microbiologyresearch.org
by
IP: 88.99.165.207
On: Wed, 10 May 2017 17:55:41
1782
K.-I.T A C H I B A N A A N D OTHERS
during the formation of spheroplasts. Journal of
JACOBSON,
G. R. & ROSENBUSCH,
J. P. (1976).
Biological Chemistry 240, 3685-3692.
Abundance and membrane association of elongation
J. 0. (1982). MembraneNIELSEN,J. B. K. & LAMPEN,
factor Tu in E. coli. Nature, London 261, 23-26.
bound penicillinases in gram-positive bacteria. JourKREIL,G. (1981). Transfer of proteins across memnal of Biological Chemistry 257, 44904495.
branes. Annual Review of Biochemistry 50, 317-348.
J., KEMPE,T. & MESSING,J. (1983)
LAEMMLI,
U. K. (1970). Cleavage of structural proteins NORRANDER,
Construction of improved M13 vectors using oligoduring the assembly of the head of bacteriophage T4.
deoxynucleotide-directed mutagenesis. Gene 26,
Nature, London 227, 680-685.
MCLAUGHLIN,
J. R., MURRAY,
C. L. & RABINOWITZ, 10 1- 106.
K., YAMAZAKI,
H., TAKEICHI,Y., NAKAJ. C. (1981). Unique features in the ribosome OHMURA,
YAMA,
A., OTOZAI,K.,YAMANE,
K., YAMASAKI,M.&
binding site sequence of the Gram-positive StaphyloTAMURA,G. (1983). Nucleotide sequence of the
coccus aureus b-lactamase gene. Journal of Biological
promoter and NH2-terminal signal peptide region of
Chemistry 256, 11283-1 1291.
Bacillus subtilis a-amylase gene cloned in PUB1 10.
MAMATIS,T., FRITSCH,
E. F. & SAMBROOK,
J. (1982).
Biochemical and Biophysical Research CommunicaMolecular Cloning,a Laboratory Manual. Cold Spring
tions 112, 678-683.
Harbor, New York: Cold Spring Harbor
Om,T., SAKAMOTO,
S.,MIYOSHI,K., FWA, T., YODA,
Laboratory.
K., YAMASAKI,
M., TAMURA,G. & MIYAKE,T.
MXmLA, P. & ZALKIN,H. (1979). Membrane-bound
(1985). Synthesis and secretion of human epidermal
and soluble extracellular a-amylase from Bacillus
growth factor by Escherichia coli. Proceedings of the
subtilis. Journal of Biological Chemistry 254, 8540National Academy of Sciences of the United States of
8547.
America 82, 72 12-72 16.
MATSUZAKI,
H.,YAMANE,
K., NAGATA,
Y. & MARUO,
B. (1974). Hybrid a-amylases of Bacillus subtilis. I. PALVA,I., PETTERSSON,R. F., KALKKINEN,N.,
LEHTOVMRA,
P., SARVAS,M., SODERLUN,
D. H.,
Purification and characterization of extra cellular aTAKKINEN,
K. AND KMRRIAINEN,
L. (1981). Nucleoamylases produced by the parental strains and
tide sequence of the promoter and NH2-terminal
transformants. Biochimica et biophysica acta 365,
signal peptide region of the a-amylase gene from
235-247.
Bacillus amyloliquefaciens. Gene 15, 43-5 1.
MICHAELIS,
S. & BECKWITH,
J. (1982). Mechanism of
F., NICKLEN,S. & COULSON,A. R. (1977).
incorporation of cell envelope proteins in Escherichia SANGER,
DNA sequencing with chain-terminating inhibitors.
coli. Annual Review of Microbiology 36, 435-465.
Proceedingsof the National Academy of Sciences of the
MILLER, J. H. (1972). Experiments in Molecular
United States of America 74, 5463-5467.
Genetics, pp. 352-355. Cold Spring Harbor, New
TAKEICHI,
Y., OHMURA,
K.,N ~ Y A M A.,
A , OTOWI,
York: Cold Spring Harbor Laboratory.
K. & YAMANE,
K. (1983). Cloning of Bacillus subtilis
MIYAKE,T., Om, T., NISHIWWA,T., M m m , F.,
a-amylase structural gene in plasmid PUB1 10.
M. &TAMURA,G.
FUWA,T., YODA,K., YAMASAKI,
Agricultural and Biological Chemistry 47, 159- 16 1.
(1985). Secretion of human interferon-a induced by
WALLACE,
R.B., JOHNSON,P. F., TANAKA,
S.,SCHOLD,
using secretion vectors containing a promoter and
M., ITAKURA,
K. & ABELSON,
J. (1980). Directed
signal sequence of alkaline phosphatase gene of
deletion of a yeast transfer RNA intervening
Escherichia coli. Journal of Biochemistry 97, 1429sequence. Science 209, 1396-1400.
1436.
YAMAWCI,
H., OHMURA,
K., NAKAYAMA,
A., TAKE+
MIYOSHI,
K., MIYAKE,
T., HOZUMI,T. & ITAKURA,
K.
CHI, Y., OTOZAI,K., YAMASAKI,
M., TAMURA,
G. &
(1980). Solid phase synthesis of polynucleotides. 2.
YAMANE,
K. (1983). a-Amylase genes from an aSynthesis of polythymidylic acids by the block
amylase hyperproducing Bacillur subtilis strain.
coupling phosphotriester method. Nucleic Acids
Journal of Bacteriology 156, 327-331.
Research 8, 5473-5489.
YANG, M., GALIZZI, A. & HENNER,D. (1983).
NEIDHART,
F. C., BLOCH,P. L. &SMITH, D. F. (1974).
Nucleotide sequence of the amylase gene. Nucleic
Culture medium for enterobacteria. Journal of
Acids Research 11, 237-249.
Bacteriology 199, 736-747.
NEU, H. C. & HEPPEL,L. A. (1965). The release of
enzymes from Escherichia coli by osmotic shock and
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