Note: Descriptions are shown in the official language in which they were submitted.
6S35
BEHRING~ERKE AKTIENGESELLSCHAET 88~B 045 - Ma 738
Dr. Lp/rd
cDNA coding for placenta protein ll (PP11), the isolation
and use thereof
The invention relates to the isolation of the gene which
codes for placenta-specific protein 11 (PP11) and to the
use thereof for the preparation of PPll by genetic
manipulation.
PPll has the following properties according to the
description in EP-B10,029,191:
a) a carbohydrate content of 3.9 + 0.9%, composed of
2.6 i 0.5% hexoses, 1.0 i 0.3% hexosamines, 0.05 i
0.03~ fucose and 0.26 i 0.07% neuraminic acid;
b) a sedimentation coefficient S of 3.5 i 0.2 S;
5 c) a molecular weight, determined in an ultracentri-
fuge, of 44,300 + 6,000;
d) a molecular weight, determined in a sodium dodecyl
sulfate (SDS)-containing polyacrylamide gel, of
62,000 i 3,000;
e) an extinction coefficient E (280nm) of 13.4 i 1.0
and
f) an electrophoretic mobility in the region of the
alpha1 globulins
as well as the amino acid composition depicted in
Table 1.
Z~06S3~i
-- 2 --
Tab. l
Amino acid composition of PP11 (residues per 100 residues
in mol-~)
CV%
(Coefficient
of variation)
Lysine 6.26 7.00
Histidine 3.34 1.72
Arginine 3.31 5.60
Aspartic acid 10.75 2.43
Threonine 3.31 10.49
Serine 9.63 1.84
Glutamic acid 13.81 2.09
Proline 4.10 4.68
Glycine 6.23 6.26
Alanine 6.30 1.82
Cystine 1/2 3.37 4.53
Valine 4.53 5.40
: Methionine 1.00 26.22
Isoleucine 3.60 2.24
Leucine 6.74 1.05
Tyrosine 5.90 3.02
i Phenylalanine 6.06 2.52
Tryptophan 1.66 20.87
Since the conventional i~olation of PPll described in the
abovementioned patent is extremely laborious, the ob~ect
therefore was to isolate the gene coding for PP11 in
order to make it possible to prepare PP11 by genetic
manipulation.
Initially, polyclonal antibodies were raised with PP11
purified conventionally as described above and were used
to search for positive clones in a commercially available
: cDNA expression bank of human placenta (Genofit GmbH,
Heidelberg; HL 1008 Human Placenta gtll Placental tissue,
34 weeks old). One clone out of 1.2 million recombinant
clones reacts with the antiserum. It was called PPl1-93
- 2~0~i~35
-- 3 --
and contained an insert of about 1,800 base-pairs ~bp).
This insert had an open reading frame of about 860 bp but
had no start methionine. Subsequently the insert of
PP11-93 was labelled and used as probe in a placenta cDNA
bank in lambda gtlO prepared by u5. Two further clones
which overlap with PP11-93 were found (PP11-166 and
PP11-169), and their sequence was determined. Although
clone PP11-169 is shorter than clone PP11-93, it overlaps
the latter at the 5' end by 270 bp and has a methionine,
which must be the start methionine. Using a 5'-terminal
46mer oligonucleotide of the clone PP11-169 (5~
CTCCAACTGGGCACCATGAGGGCCTGCATCTCCCTGGTATTGGCCG), finally
the entire 5'-untranslated sequence was found in the
clone PP11-318, which shows that there are now only stop
codons in front the abovementioned presumptive start
methionine and no further methionine appears. Thus, the
complete cDNA of PPll is, including the poly(A) sequence,
2399 bp long and has an open reading frame for 369 amino
acids, which corresponds to a protein of 42,120 D. 154 bp
at the 5' end are untranslated, 1138 bp are untranslated
at the 3' end, and a 1107 bp coding sequence iB enC108ed.
The complete cDNA sequence is listed in Table 2, and the
figure shows the position of the individual clones within
the cDNA as well as the coding region. "N" therein
represents the N terminus, "C" represents the C terminus
of the coding region and "A" represents the poly(A)
sequence.
It is possible according to the invention for the coding
cDNA to be used, by means of suitable expression systems,
for expressing PPll. It is furthermore possible, by the
choice of the host, to influence the type of modification
of PPll. Thus, no glycosylation takes place in bacteria,
and that takinq place in yeast cells differs from that in
higher eukaryotic cells. The expression of PPll takes
place particularly advantageously in E.coli with the
expression vector pTrc 99A or pTrc 99C (see Example 11).
It has furthermore been found that PPll, preferably
prepared as above by genetic manipulation in E.coli or
i1653S
-- 4 --
yeast, can be employed for the treatment of coagulation
disorders, disorders of the complement system or in
connection with disorders of fibrinolysis, because it has
an amidolytic activity.
It is possible, knowing the amino acid sequence of PPll,
to prepare, by conventional methods of genetic manipu-
lation, amino acid partial sequences which act as anti-
gens for the preparation of polyclonal or monoclonal
antibodies. Antibodies of these types can be employed not
only for diagnostic purposes but also for the preparation
of antibody columns. PP11 can thus be removed from
solutions which contain it in addition to other proteins.
It i8 also possible with the aid of the cDNA or parts
thereof to isolate in a straightforward manner from a
genomic bank the genomic clone coding for PP11, with
whose aid not only is expression in eukaryotic cells
facilitated but also further diagnostic conclusions can
be drawn.
The invention is furthermore defined in the patent claims
and detailed in the examples which follow.
Where not explained in the text, the following abbrevi-
ations are used:
EDTA = Sodium ethylenediaminetetraacetate
SDS = Sodium dodecyl sulfate
DTT = Dithiothreitol
BSA = Bovine serum albumin
Examples:
1. Isolation of RNA from human placenta
RNA was obtained from mature human placenta (method of
Chirgwin et al., Biochemistry 18 (1079) 5294-5299)).
About 10 g of placenta tissue were ground in a mortar
under liquid nitrogen, suspended in 80 ml of 4 M guani-
dinium thiocyanate containing 0.1 M mercaptoethanol and
ZC~ 535
-- 5 --
treated in a homogenizer (Ultraturrax) at 20,000 rpm for
90 sec. The lysate was centrifuged at 7,000 rpm for 15
min (Sorvall GSA rotor) and the supernatant was precipi-
tated with 2 ml of 1 M acetic acid and 60 ml of ethanol
abs. at -20C overnight. After sedimentation at 6,000 rpm
and -10C for 10 min, the nucleic acids were completely
dissolved in 40 ml of 7.5 M guanidinium hydrochloride (pH
7.0) and precipitated with a mixture of 1 ml of 1 M
acetic acid and 20 ml of ethanol abs. To remove the DNA,
the precipitation was repeated once more with each of the
volumes halved. The RNA was dissolved in 12 ml of H2O,
precipitated with a mixture of 1.2 ml of 4 M potassium
acetate and 24 ml of ethanol abs., sedimented and finally
taken up again in 10 ml of H2O (1 ml per g of tissue).
2. Obtaining poly(A~-containing placenta mRNA
The placenta RNA was fractionated by oligo(dT)-cellulose
chromatography (Aviv and ~eder, Proc. Natl. Acad. Sci.
~SA 69 (1973) 1408-1412) in 2 ml Pasteur pipettes in LiCl
in order to obtain poly(A)-containing mRNA. About 5 mg of
placenta RNA in buffer 1 (500 mN LiCl, 20 mM Tris (pH
7.S), 1 mM EDTA, 0.1% SDS) were loaded onto the column.
Whereas the poly(A)+-RNA was bound to oligo(dT)-cellulose,
it was possible to elute the poly(A) -RNA again. After a
washing step with buffer 2 (100 mN LiCl, 29 mM Tris (pH
7.5), 1 mM EDTA, 0.1% SDS), the poly(A)t-RNA (placenta
mRNA) was eluted from the column with buffer 3 (5 mM Tris
(pH 7.5), 1 mm EDTA, 0.05% SDS).
For further purification, the poly(A)+-RNA was ad~usted
to buffer 1 and again chromatographed on oligo(dT)-
cellulose. The yield of placenta poly(A)+-RNA after this
second purification step was about 4% of the RNA
employed.
3. Synthesis of cDNA from human placenta (placenta
cDNA) and double-stranded cDNA (dsDNA)
.
2~G535
Poly(A)-containing placenta mRNA was checked for in
tegrity before the cDNA synthesis in a 1.5% agarose gel.
Then 4 ~g of placenta mRNA were dissolved in 65.5 ~l of
H20, denatured at 70C for 10 min and cooled on ice.
The cDNA was synthesized in a 100 ~l mixture after
addition of 20 ~l of RTl buffer (250 mM Tris (pH 8.2) at
42C, 250mM KCl, 30mM MgCl2), 2.5 ~l of 20 mM dNTP (i.e.
all four deoxynucleoside triphosphates), 1 ~l of oli-
gotdT) of 1 ~g/ml, 1 ~l of 1 M DTT, 2 ~l of RNAsin and
8 ~l of reverse transcriptase (24 U/~l) at 42C for 90
min. Double-stranded cDNA (dsDNA) was synthesized by the
method of Gubler and Hoffmann (Gene 25 (1983) 263-269).
The synthesis was carried out immediately after the cDNA
synthesis by adding 305.5 ~l of ~2~ 80 ~l of RT2 buffer
(100 mM Tris (pH 7.5), 25 mM MgCl2, 500 mM KCl, 50 mM DTT,
250 ~g/ml BSA), 2 ~l of RNase H (2 U/~l), 2.5 ~l of
E.coli DNA ligase (5 U/~l), 5 ~1 of 15 mN ~-NAD, and 5 ~1
of DNA polymera~e I (5 U/~l) and incubation at 15C for
5 h. The reaction was stopped by heat inactivation
(70C, 30 min).
55 ~1 of 250 ~M dNTP, 55 ~l of 10 mM Tris (pH 7.5), 10 mM
MgCl2, 10 ~g/ml BSA, 3 ~l of T4 DNA polymerase I (1 U/~l),
2 ~l of RNase H (2 U/~l) and 2 ~l of RNa~e A (2 ~g/ml)
were added to the reaction mixture, which was then
incubated at 37C for a further 30 min in order to ensure
that the synthesis of the second DNA strand was complete
(repair reaction).
4. Ligation of EcoRI linkers to the dsDNA and opening
of the linkers
To set up a placenta cDNA bank, the dsDNA wa~ provided
with EcoRI ends in order to be able to ligate it into the
EcoRI cleavage 6ite of the phage vector lambda gtlO
(T. Maniatis et al. (1982), Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor). For this purpose,
006535
the dsDNA was
a) treated with EcoRI methylase in order to protect
internal EcoRI cleavage sites in the dsDNA,
b) provided with EcoRI linkers which
c) were then opened with EcoRI.
Re a):
The methylase reaction of the dsDNA was carried out
immediately following the repair reaction after addition
of 25 ~1 of 500 mN EDTA (pH 8.0), 60 ~1 of methylase
buffer (100 mN NaOAc (pH 5.2), 2 mg of S-adenosyl-L-
methionine) and 2 ~1 of EcoRI methylase (20 U/~l) by
incubation at 37C for 30 min. The reaction mixture was
extracted with phenol, and the dsDNA was precipitated
with 60 ~1 of 4M NaOAc and 1300 ~1 of ethanol. The dsDNA
was washed twice with 70~ ethanol, extracted by shaking
once with ether and dried.
Re b):
The EcoRI-methylated dsDNA was dissolved in 88 ~1 of H20
and, after addition of 10 ~1 of ligase buffer (500 mM
Tris (pH 7.4), 100 mM MgCl2, 100 mM DTT, 10 mM spermidine,
10mM ATP, 1 mg/ml BSA), 1 ~1 of T4 DNA ligase (10 U/~l),
ligated with 1 ~1 of EcoRI linkers (0.5 ~g/~l) (pGGAATTCC
and pAGAATTCT) at 15C overnight.
Re c):
The volume of the ligase mixture was adjusted to 120 ~1
with 6 ~1 of H2O, 12 ~1 of 10 x EcoRI buffer and 2 ~1 of
EcoRI (120 U/~l). The EcoRI digestion was carried out at
37C for 2 h.
5. Removal of unbound linkers on potassium acetate
, . .- - .
~ ~0~)~s3s
-- 8 --
gradients and selection of the dsDNA for size
To remove all unbound EcoRI linkers from the dsDN~, the
EcoRI reaction mixture was loaded in toto onto a potas-
sium acetate gradient (5-20% XAc, l mM EDTA, l ~l/ml
ethidium bromide) and centrifuged at 50,000 rpm and 20C
for 3 h (Beckman SW 65 rotor).
The gradient was fractionated from below in such a way
that the volume of the first five fractions was 500 ~il
and that of the remainder was 100 ~1. The fractions were
precipitated with 0.01 volume of acrylamide (2 mg/ml) and
2.5 volumes of ethanol, washed once with 70~ strength
ethanol, dried and taken up in 5 ~il of H20 in each case.
To determine the ~ize of the dsDNA, 1 ~l of each fraction
was analyzed in a 1.5% agarose gel. In addition, the
quantity of dsDNA was determined using l ~il of each
fraction.
Fractions with dsDNA over 1,000 bp were combined and the
sample was concentrated until the final concentration was
27 ~g/ml.
6. Incorporation of the dsDNA into the phage vector
lambda gtlO and in vitro packaging reaction
The dsDNA was incorporated into the EcoRI cleavage site
of the phage vector lambda gtlO (Vector Cloning Systems,
San Diego, CA) in a 4 ~il ligase mixture: 2 ~il of dsDNA,
1 ~l of lambda gtlO x EcoRI (1 ~g/ml), 0.4 ~l of ligase
buffer, 0.5 ~il of H~O, 0.1 ~il of T4 DNA ligase. The
mixture was incubated at 15C for 4 h.
To establish the placenta cDNA bank in the phage vector
lambda gtlO, an in vitro packaging reaction of the ligase
mixture was subsequently carried out with the
lambdalysogenic cell extracts E.coli NS 428 and NS 433 at
room temperature for 2 h (Vector Cloning Systems, San
, ,
2~S35
g
Diego, CA; Enquist and Sternberg, Methods in Enzymology
68, (1979), 281-298). The reaction was stopped with
500 ~1 of suspending medium (SM: 0.1 M NaCl, 8 mM MgSO4,
50 mM Tris (pH 7.5), 0.01~ gelatin) and 2 drops of
chloroform.
7. Determination of the titer and analysis of the
placenta cDNA bank
The number of plaque-forming units (PFU) of the placenta
cDNA bank was determined using competent cells of the
E.coli K 12 strain C600 HFL: it was 1 x 106 PFU. About
80~ of all the phages contained DNA inserts which were
larger than 1,000 base-pairs.
8. Screening of an expression cDNA bank from human
placenta with anti-PP11 antibodies
An expression cDNA bank in phage lambda gtll from Genofit
(loc. cit.) was plated out at a density of about
30,000 PPU per agar plate (13.5 cm diameter). For this,
competent cells of the E.coli strain ylO90 (ATCC 37197)
(R.A. Young and R.W. Davis, Science Vol. 222,
778-782/1983) were infected with the phages at 37-C for
30 min and then plated out in the top agar on L broth
plates. The plates were incubated at 42C for 4 h and
then each was covered with a dry nitrocellulose filter
(Schleicher and Schuell, BA 85, Ref. No. 401124). The
filters had previously been saturated with 10 mM IPTG in
water. The plate with the filter~ was sub~ected to
renewed incubation at 37C for 4 h. Before the filter~
were removed again, the filters and plate were marked
simultaneously with a needle dipped in carbon black. The
filters were then incubated in TBST (10 mM Tris-HCl, pH
8.0, 150 mN NaCl, 0.05% Tween 20 and 5% skim milk powder)
at 4C overnight. The filters were subsequently washed
three more times for 10 min in TBST at room temperature
and then incubated with rabbit anti-Pill antibodies in
15 ml of TBST per filter at room temperature for 1 h.
, .
G535
-- 10 --
(The solution of the antibodies had previously been
diluted 1:200 and saturated with non-recombinant lambda
gtll lysed E.coli cells on nitrocellulose filters for 1
h). After the filters had been incubated with the prLmary
antibody they were washed 4 x 10 min with T~ST. The
filters were then incubated, shaking for 1 h, with the
~econdary anti-rabbit antibody which was con~ugated to
alkaline phosphatase (from Promega, USA - marketed by
Atlanta, Heidelberg) and had previously been diluted
1:5,000 in TBST. The filters were then washed again 4 x
10 min with TBST. Finally, the color reaction was carried
out in order to visualize the PPll-positive clones to
which both primary and secondary antibodies were bound,
by reaction of the alkaline phosphatase and a color
reagent (ProtoBlot system from Protogen). For each color
reaction, 15 ml of AP buffer (100 mN Tris-HCl, pH 9.5,
100 mM NaCl, 5 mM MgCl2) were mixed with 99 ~1 of NBT
(nitro blue tetrazolium) substrate (50 mg/ml in 70%
dimethylformamide) and 49.5 ~1 of BCIP (5-bromo-4-chloro-
3-indolyl phosphate) substrate (50 mg/ml in 70~ dimethyl-
formamide) for one nitrocellulose filter. The filters
were agitated in the stain solution in the dark for about
20 min to 1 h until positive plaques showed an adequate
blue stain. The color reaction was stopped by immersing
the filters in a stop solution (20 mM Tris-HCl, pH 8.0
and 5 mM EDTA).
Positive signals were assigned to the plaques on the
corresponding agar plate. The plaque~ were removed by
stabbing with a Pasteur pipette, resuspended in 1 ml of
SM buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2) and
singled out to obtain a single positive plaque. From the
expression gene bank used here there wa~ found a clone
(PP11-93) which, however, did not contain the complete
gene but, with the total length of the insert being about
1800 bp, had an open reading frame - without start
methionine - of 860 bp.
9 a) Preparation of a PPll-specific probe
2~535
; The cDNA was isolated by known methods from the PPll-g3
clone and labelled with 32p as follows.
10 ng of DNA were labelled using the Multiprime DNA
labelling system (from Amersham, Braunschweig) with
Klenow polymerase in the presence of alpha-32P-dCTP
(35 ~Ci/25 ~1 of reaction mixture were employed). The DNA
probe had a specific activity of 330 MBq/pmol.
9 b) Screening of the placenta cDNA bank, prepared by us
as in 1. to 7., with PP11-specific probes
For this, 3 x 104 PFU were plated out with cells of the
E.coli K 12 strain C 600 HFL in soft agar on 13.5 cm
Petri dishes and incubated at 37DC for 6 h. Lysis was not
yet complete at this time. The plates were incubated in
a refrigerator overnight, and the pha~es were transferred
onto nitrocellulose filters ( Schleicher & SchUll, BA 85,
Ref. No. 401124) (duplicates). Nitrocellulose filters and
Petri dishes were marked with an in~ection needle in
order to allow subsequent assignment. The Petri dishes
were stored in a cold room while the nitrocellulose
filters were being processed. The DNA present on the
nitrocellulose filters was denatured by placing the
filters for 5 min on filter paper (Whatman M3) impreg-
nated with 1.5 M NaCl, 0.5 M NaOH. The filters were
subsequently renatured in the same way with 1.5 M NaCl,
0.5 M Tris (pH 8.0) and washed with 2 x SSPE (0.36 M
NaCl, 16 mM NaOH, 20 mM NaH2PO4, 2 mM EDTA). The filters
were then dried in vacuo at 80 C for 2 h. The filters
were prehybridized at 65C for 4 h (prehybridization
solution: 0.6 M NaCl, 0.06 M Tris (pH 8.3), 6 mM EDTA,
0.2% non-ionic synthetic sucrose polymer, (Ficoll), 0.2%
polyvinylpyrrolidone 40, 0.2% BSA, 0.1% SDS, 50 ~g/ml
denatured herring sperm DNA). The filter~ were finally
incubated overnight with addition of 100,000 - 200,000 Bq
of the labelled oligonucleotide/ml of hybridization
solution (as prehybridization solution but without
.. - .
-,
2~ ;35
- 12 -
herring sperm DNA) in beakers or in sealed polyethylenefilms, shaking gently. The hybridization temperature was
65C. The nitrocellulose filters were washed with 6 x
SSC, 0.05 M sodium pyrophosphate at room temperature for
one hour and at the particular hybridization temperature
for a further hour. The filters were dried and auto-
radiographed overnight. X-ray film signals which occurred
on both duplicates were assigned to the Petri dish, and
the region (about 50 plaques) was removed by stabbing
with the wide end of a Pasteur pipette, and the phages
were resuspended in 1 ml of SM buffer. Positive phages
were singled out in three cycles until a single clone was
obtained.
In total, 1 x 106 PFU of the placenta cDNA bank were
examined. 2 further signals were identified on duplicate
filters (clones PP11-166 and PP11-169). Finally, with the
aid of the 46mer from PP11-169, which is mentioned above
(p.3), the clone PP11-318 which covers the entire 5' end
of the PP11 cDNA was found. These 4 clones together carry
a PP11-encoding sequence as depicted in Tab. 2 and the
figure.
10. DNA sequence analysis
The phage clones (clones PP11-93, PP11-166, PP11-169 and
PP11-318) were multiplied and the DNA of each was ex-
tracted. The relevant EcoRI fragment was isolated andligated into the EcoRI site of the Bluescript M13 vector
(Stratagene, San Diego, CA, USA) for restriction analyses
and sequence analyses by the enzymatic dideoxy method of
Sanger. The sequence has an open reading frame and codes
for a protein with a maximum of 369 amino acids. Thus,
there are 154 bp not translated at the 5' end, and these
are followed by 1107 bp of coding sequence and finally
1138 bp of untranslated sequence at the 3' end.
11. Expression of PPll in bacterial cells
200G535
_ 13 -
a) Expression of ~ PPll fu~ion protein
pTrc99C (E. Amann et al., Gene 69 tl988) 301-315) was
completely digested with EcoRI and XbaI, and the fragment
4149 base-pairs in size was isolated by gel electro-
phoresis. The PP11 clone lambda gtll-169 was digested
with EcoRI, and the EcoRI insert which is 1785 base-pairs
in size, including the linker portion, was likewise
\ isolated. The PPll cDNA of the clone lambda gtll-169
starts at position 140 in Figure ~. The resulting
fragment was then cut with XbaI, and the EcoRI-XbaI
fragment which is 1339 base-pairs in ~ize was isolated
and ligated to the pTrc99C vector fragment described
above. The 5488bp plasmid pTrc99C-PPll obtained in this
way is able to induce the synthesis of an approximately
42 kD protein in Escherichia coli cells. This protein can
be specifically immunoprecipitated with aid of a mono-
specific rabbit anti-PPll antiserum which has been raised
by immunization with PPll isolated from human placenta.
PPll expression in Escherichia coli cells is additionally
detected by Western blot analyses using the above serum.
In this experiment, only the extract from IPTG-induced
cells containing pTrc99C-PPll plasmid reacted, once again
a protein band of about 42 kD being specifically visual-
ized. Escherichia coli control extracts without plasmid,
and extracts containing pTrc99C-PPll but not induced with
IPTG, did not react with the abovementioned anti-PPll
antiserum. The PPll fusion protein described by the
pla~mid construction generated hereinbefore has the
following N-terminal amino acid sequence, defined by the
following nucleotide seguence:
Vector/linker / 5' UT region / PPll
1 2 3 4 5 6 7 8 9 1 2 3
Met Gly Asn Ser Leu Gln Leu Gly Thr Met Arg Ala
..CC ATG GGG AAT TCT CTC CAA CTG GGC ACC ATG AGC GCC...
The PPll fusion protein defined by this construction
carries, in addition to the complete PPlI amino acid
sequence encoded by the PPll cDNA, nine amino acids in
.
` ' ' '' '
. ,
ZC~ 53~
front of the N-terminus: four vector-encoded amino acids
plus five amino acids specified by the 5' untranslated
region occurring in the PP11 cDNA. As a check, the
construction indicated hereinbefore was likewise carried
out with the expression vectors pTrc99A and pTrc99B
(German Patent Application P 38 19 463.5 and Amann et al.
(1988) Gene 69, ~01-315). These vectors differ from
pTrc99C only by 2 base-pairs (pTrc99A) and 1 base-pair
(pTrc99B), which cause shifts in the translation reading
frame. As expected, neither pTrc99A-PPll nor pTrc99B-PP11
was able to induce the synthesis of PPll proteins react-
ing with anti-PPll antisera.
It was found, surprisingly, that the PPll protein induced
by plasmid pTrc99C-PP11 is processed in E~cherichia coli.
The signal sequence which in nature precedes the mature
PPll protein (also called leader sequence) is recognized
by E.coli cells and cleaved by the signal peptidase. It
is possible to conclude this from the finding that the
PP11 protein detected in the periplasmic fraction after
fractionation of cell extracts is smaller than in the
membrane-associated fraction, with the observed molecular
weight difference corresponding to the expected dif-
ference after elimination of the signal sequence.
b) Expression of the mature unfused protein:
To express the mature PP11 protein lacking the naturally
occurring signal sequence, the EcoRI-XbaI fragment which
is 1339 base-pairs long and is described hereinbqfore was
ligated to the mutagenesis vector pMa5-8 which had been
digested with the same restriction enzymes. The muta-
genesis vector pMa5-8 carries a cloning polylinker region
and the origin of replication of the single-stranded
bacteriophage F1, in addition to a bacterial origin of
replication and an antibiotic-resistance marker. The
resulting plasmid pMa5-8-PP11 was used to prepare a
single strand. The single strand of the cloned PP11 cDNA
obtained in this way was then isolated by known methods
6~35
_ 15 -
and subjected to the published gapped duplex mutagenesis
protocol (Kramer et al. (1984) Nucl. Acids. Res. 12,
9441-9456), using the following oligodeoxynucleotide,
5~ TTTGTGGTCCTCCATGGAGGCCAGGCCACA 3'
A clone which had the desired NcoI mutation (creation of
a new NcoI site, which was not present in the originally
isolated cDNA, immediately in front of the coding se-
quence for the mature PPll protein) was identified by
appropriate restriction analysis and called pMa5-8-PPll-
NcoI. Following partial NcoI and complete XbaI digestion,the NcoI-XbaI fragment which is 1268 bp in size was
isolated from this plasmid and ligated into the vector
pTrc99A which had been cut in the same way. The resulting
plasmid contains 5412 bp and, after induction of the trc
promoter with IPTG, expresses the mature unfused PPll
protein with a molecular weight of about 42 kD, which in
turn specifically reacts with anti-PP11 antisera, as
already described hereinbefore.
12. Amidolytic activity of PP11
PP11 was shown to have protease activity in the following
colorimetric assay mixture. Periplasmic or cytoplasmic
fractions of E.coli strain RB791 (R. Brent and M. Ptashne
(1981) Proc.Natl.Acad.Sci ~SA 78, 4204-4208) transformed
with pTrc99C or pTrc99C-PP11 were measured in an optical
assay using the chromogenic substrate Chromozym0 TH
(Boehringer Mannheim GmbH). 400 ~1 of triethanolamine
buffer and 100 ~1 of substrate were mixed with 300 ~1 of
each E.coli extract, these extracts having been diluted
to a protein concentration of 16 mg/ml. The reaction
cuvette was incubated at 37C, and the change in absorp-
tion per minute at 405 nm was recorded. In some experi-
ments the E.coli extract and control extract were pre-
incubated with a rabbit anti-PP11-antiserum, and the
amidolytic activity of the antiserum was subtracted in
the calculation of the amidolytic activities of the
extracts. At the same total protein concentration, the
PP11-containing E.coli extract brought about a larger
.
~0~6535
- 16 -
increase in absorption (~A = 0.04 per minute) than the
control (~A = 0.024 per minute), corresponding to
32 mU/ml and 19.2 mU/ml respectively. The amidolytic
activity of PP11 was almost completely inhibited by pre-
incubation with anti-PP11 antiserum. Furthermore, the
cytoplasmic unprocessed PP11 with the molecular weight of
45 kD did not have this amidolytic activity. Since the
amidolytic activity is inhibited by diisopropyl fluoro-
phosphate, PPll is probably a serine proiease.
-` 2~535
- 17 -
CTTCCTGAAAGGATCTGGAGACACCAGCTCCACAAGTCCTGGTGTCTTTAAAAGGATCAG
110
CTTGAGGAATAAGGCTCGTCTGAGAGCTGTGACATTCATCTGACTCTAGTGAAAGTCCAA
130 150 170CAGCCACTCCCTTTTTGGCCTCCAACTGGGCACCATGAGGGCCTGCATCTCCCTGGTATT
M R A C I S L V L
190 210 230
GGCCGTGCTGTGTGGCCTGGCCTGGGCTGAGGACCACAAAGAGTCAGAGCCATTGCCACA
A V L C G L A W A E D H R E S E P L P Q
250 270 290
GCTGGAGGAAGAGACAGAAGAGGCCCTCGCCAGCAACTTGTACTCGGCACCCACCTCCTG
L E E E T E E A L A S N L Y S A P T S C
310 330 350
CCAGGGCCGCTGCTACGAAGCCTTTGACAAGCACCACCAATGTCACTGCAATGCCCGCTG
Q G R C Y E A F D K H H Q C H C N A R C
370 390 410
CCAAGAGTTTGGGAACTGCTGCAAGGATTTTGAGAGCCTGTGTAGTGACCACGAGGTCTC
Q E F G N C C K D F E S L C S D H E V S
430 450 470
CCACAGCAGTGATGCCATAACAAAAGAGGAGATTCAGAGCATCTCTGAGAAGATCTACAG
H S S D A I T K E E I Q S I S E K I Y R
490 510 530
GGCAGACACCAACAAAGCCCAGAAGGAAGACATCGTTCTCAATAGCCAAAACTGCATCTC
A D T N K A Q K E D I V L N S Q N C I S
550 570 590
CCCGTCAGAGACCAGAAACCAAGTGGATCGCTGCCCAAAGCCACTCTTCACTTATGTCAA
P S E T R N Q V D R C P R P L F T Y V N
610 630 650
TGAGAAGCTGTTCTCCAAGCCCACCTATGCAGCCTTCATCAACCTCCTCAACAACTACCA
E K L F S R P T Y A A F I N L L N N Y Q
670 690 710
GCGGGCAACAGGCCATGGGGAGCACTTCAGTGCCCAGGAGCTGGCCGAGCAGGACGCCTT
; R A T G H G E H F S A Q E L A E Q D A F
730 750 770
CCTCAGAGAGATCATGAAGACAGCAGTCATGAAGGAGCTCTACAGCTTCCTCCATCACCA
L R E I M K T A V M R E L Y S F L H H Q
790 810 B30
GAATCGCTATGGCTCAGAGCAAGAGTTTGTCGATGACTTGAAGAACATGTGGTTTGGGCT
N R Y G S E Q E F V D D L K N M W F G L
Tab. 2
.
-
2C~)6535
- 18 -
850 870 B90
CTATTCGAGAGGCAATGAAGAGGGGGACTCGAGTGGCTTTGAACATGTCTTCTCAGGTGA
Y S R G N E E G D S S G F E H V F S G E
910 930 950
GGTAAAAAAAGGCAAGGTTACTGGCTTCCATAACTGGATCCGCTTCTACCTGGAGGAGAA
V K K G K V T G F H N W I R F Y L E E K
970 990 1010
GGAGGGTCTGGTTGACTATTACAGTCACATCTACGATGGGCCTTGGGATTCTTACCCCGA
E G L V D Y Y S H I Y D G P W D S Y P D
1030 1050 1070
TGTGCTGGCAATGCAGTTCAACTGGGACGGCTACTATAAGGAAGTGGGCTCTGCTTTCAT
V L A M Q F N W D G Y Y K E V G S A F
1090 1110 1130
CGGCAGCAGCCCTGAGTTTGAGTTTGCACTCTACTCCCTGTGCTTCATCGCCAGGCCAGG
G S S P E F E F A L Y S L C F I A R P G
1150 1170 1190
CAAAGTGTGCCAGTTAAGCCTGGGAGGATATCCCTTAGCTGTCCGGACATATACCTGGGA
K V C Q L S L G G Y P L A V R T Y T W D
1210 1230 1250
CAAGTCCACCTATGGGAATGGCAAGAAGTACATCGCCACAGCCTACATAGTGTCTTCCAC
K S T Y G N G K K Y I A T A Y I V S S T
1270 1290 1310
CTAATAGAACTTCGAGCCAGAAAGGGGCATGAGGGCTCTTGCGAGACTGAAGTGCTATCT
1330 1350 1370
TCTCTGGACTAGAGAGAAGAGGGAGAGGACTGGAAGGGATCACCAAATCTCAAAGCAATG
1390 1410 1430
AGAAGCATTCCTAAATCCCAAAGTGCCCACATGGGAAAGAGATAAAATGTACAAATTAGA
1450 1470 1490
AAAATGTGGATAAACAGTCAAACCTTTATCCTCTAGAATTTTGGCAATGTTGACTAAGAA
1510 1530 1550
ACAGAGTCCAAGCAGAGAAGGTAGGAACCCTCCATAGCTCTCTGCCCTGATGTGTGGGGG
1570 1590 1610
AACTAGGAAGAAGTCCTTTGACCTCACCAGGCCTCATGCTTCCCTTTAATGTAAAGGGAA
1630 1650 1670
GGGGTTTGCCCACTTTCCTCTTTTTGGGGTTGGTGAGAGGGCAAACCCTGATATTTTTAC
1690 1710 1730
TGTGAAGGTGTTTTCAGTTGTTCTTAGGAAGAACAGCTGATAGAAATTCAAGATTACTAT
1750 1770 1790
AATGGCTGTTATTATACACAGCTCTGTAAACTACCACTCAGCCCTGTGTTGGGGTCCTCA
Tab. 2 continued
~)6S:~S
-- 19 --
1810 1830 1850
AAGAAGTAAGGCCACAGTAATCAAGCAAGGGCCTTTGGTTTTTTCCAGAGTTAGATCCTC
1870 1890 1910
TCAGAACAGAGTCTGGGAGAACTCCAATGCTGAATGGAGAAGGGTAATAGGTTGGTTGCA
1930 1950 1970
GTGAATGGGCTGGGGGTGGGGTGGCCTTCTCCAGGCCTGAGTGTTTTTGTGTCCAGCTCA
1990 2010 2030
GTATCTGCAACAAGAAGTTTCCCACTTGTGGATGTTTAGTGCAGCCACAGACTTGTATTT
2050 2070 2090
TGATCCCCAATTTTTTTTGAAAGAGTTCTCCTCATAGGAGGATGATTCAGCATCAGAAGA
2110 2130 2150
AGAAGGAACCCATAGCTTGGTGTCATTAACATAATTATTTTAAGCCTTATCCAGCAGCCA
2170 2190 2210
TAATTTGAATAACTCTACGAGACCAGAGAGACTGTAGTTCCCTATTTTAACCTCAATTAT
2Z30 2250 2270
GCATTTGTCCCCAACCCCACTGAGAACTAAATGCTGTACCACAGAGCCGGGTGTGAACTA
2290 2310 2330
TGGTTTAGAAGGTTcAAGTTTccAATTAAAGTcATTGAAGAb8-~uu4~uu~u~AAAA~A
2350 2370 2390
Tab. 2 continued
