U.S. patent number 4,711,843 [Application Number 06/461,248] was granted by the patent office on 1987-12-08 for method and vector organism for controlled accumulation of cloned heterologous gene products in bacillus subtilis.
This patent grant is currently assigned to Cetus Corporation. Invention is credited to Shing Chang.
United States Patent |
4,711,843 |
Chang |
December 8, 1987 |
Method and vector organism for controlled accumulation of cloned
heterologous gene products in Bacillus subtilis
Abstract
A method and a cloning vector are described for the controlled
accumulation of cloned heterologous gene products in Bacillus
subtilis. The cloning vector is capable of being replicated in B.
subtilis and includes the heterologous gene located and oriented
such as to be under the control of an operator, promoter, and
ribosomal binding site sequence. The gene codes for a protein which
is under the control of a transport mechanism by which the protein
is secreted by the B. subtilis. The gene product is recovered from
the growth medium for the B. subtilis. The cloning vector is also
capable of similar use in other bacteria such as E. coli.
Inventors: |
Chang; Shing (Hercules,
CA) |
Assignee: |
Cetus Corporation (Emeryville,
CA)
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Family
ID: |
26916149 |
Appl.
No.: |
06/461,248 |
Filed: |
January 26, 1983 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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221800 |
Dec 31, 1980 |
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128537 |
Mar 10, 1980 |
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Current U.S.
Class: |
435/69.1;
435/231; 435/243; 435/252.31; 435/252.33; 435/320.1; 435/485;
435/69.4; 435/69.7; 435/839; 435/849; 435/91.41; 536/23.1;
536/23.2; 930/200; 930/240; 930/300 |
Current CPC
Class: |
C12N
15/67 (20130101); C12N 9/86 (20130101); C07K
2319/02 (20130101); C07K 2319/036 (20130101); C07K
2319/61 (20130101); Y10S 930/30 (20130101); Y10S
435/849 (20130101); Y10S 435/839 (20130101) |
Current International
Class: |
C12N
9/86 (20060101); C12N 9/78 (20060101); C12N
15/67 (20060101); C12P 021/00 (); C12P 021/02 ();
C12P 021/04 (); C12P 019/34 (); C12N 015/00 (); C12N
009/86 (); C12N 001/00 (); C12N 001/20 (); C12R
001/125 (); C12R 001/19 (); C07H 021/04 () |
Field of
Search: |
;435/68,70,91,172.3,253,839,243,231,172.1,849,71
;935/29,38,39,41,45,47,72,74,48,52,73 ;536/27 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Smith et al., "Extracellular Labeling of Growing Secreted
Polypeptide Chains in Bacillus subtilis with Diazoiodosulfanilic
Acid", Biochemistry 18: 198 (1979). .
Davies et al., "Plasmid-Determined Resistance to Antimicrobial
Agents", Annual Rev. Microbiol., vol. 32, pp. 469, 480, 481, and
482 (1978). .
Chang et al., "Expression of Eukaryotic Genes in B. subtilis Using
Signals of penP", in Molecular Cloning and Gene Regulation in
Bacilli, Ganesan et al. (ed.), 1982, Academic Press, New York, pp.
159-169. .
Genes, Lewin, John Wiley & Sons, New York, 1983, p. 673. .
Davis et al.: Nature 283, 433 (1980). .
Taniguchi et al., Proc. Japan. Acad. 55, Ser. B, 464 (1979). .
Gray and Chang, J. Bacteriol., 145:422-428 (1981). .
Hori and Osawa, PNAs (USA) 76:381-385 (1979). .
Lovett et al., Methods in Enzymology, vol. 68:342 (1979). .
Rubin et al., Chem. Abstr. 93, 14613p (1980). .
Gryczan et al., Molec. Gen. Genet. 177:459 (1980). .
Gryczan et al., J. Bacteriol. 141:246 (1980). .
Young et al., Genetic Engineering, Chakrabarty (ed.) CRC Press,
1978, pp. 145-147. .
Keggins et al., Proc. Natl. Acad. Sci. USA 75:1423 (1978). .
Erlich Proc. Natl. Acad. Sci. USA 74:1680 (1977). .
Erlich Proc. Natl. Acad. Sci. USA 75:1433 (1978). .
Gryczan et al., Proc. Natl. Acad. Sci. USA 75:1428 (1978). .
Duncan et al., Chem. Abstr. 86:185728k (1977). .
Kreft et al., Chem. Abstr. 89:103524r (1978)..
|
Primary Examiner: Martinell; James
Attorney, Agent or Firm: Fineman; Elliott L. McLaughlin;
Jane Meyer; Virginia
Parent Case Text
This is a continuation of application Ser. No. 221,800, filed Dec.
31, 1980, now abandoned which is a continuation-in-part of
application Ser. No. 128,537, filed Mar. 10, 1980, now abandoned.
Claims
What is claimed is:
1. A method for producing a predetermined protein comprising
providing growth conditions in a growth medium for Gram-positive
bacteria containing hybrid cloning vectors, said vectors having a
gene therein for said predetermined protein, said predetermined
protein being non-indigenous to the Gram-positive bacteria, said
gene being located and oriented in said vector such as to be under
the control of an operator, promoter, and ribosomal binding site
sequence, said predetermined protein being under the control of a
transport mechanism comprising a portion of a Bacillus secretion
leader sequence that functions for secretion by which said
predetermined protein is secreted by said Gram-positive bacteria,
culturing said bacteria under conditions suitable for the
production and secretion of said predetermined protein, and
recovering said predetermined protein from said growth medium.
2. A method according to claim 1 wherein said bacteria are Bacillus
subtilis.
3. A method according to claim 1 wherein said gene for said
predetermined protein is under the control of the beta-lactamase
operator, promoter, and ribosomal binding site sequence.
4. A method according to claim 1 wherein said predetermined protein
is beta-lactamase.
5. A method according to claim 1 wherein said predetermined protein
is a mammalian protein.
6. A method according to claim 1 wherein said predetermined protein
is a mammalian hormone.
7. A method according to claim 1 wherein said transport mechanism
is provided for by a portion of the Bacillus licheniformis
.beta.-lactamase secretion leader that functions for secretion.
8. A method according to claim 1 wherein said transport mechanism
provided for by a functional portion of a Bacillus secretion leader
sequence is a portion of the .alpha.-amylase gene.
9. A method for producing a predetermined protein non-indigenous to
Bacillus subtilis comprising providing growth conditions in a
growth medium for Bacillus subtilis bacteria containing hybrid
cloning vectors, said vectors having a gene therein for said
predetermined protein, said gene being located and oriented in said
vector such as to be under the control of an operator, promoter,
ribosomal binding site sequence, and a transport mechanism
comprising a portion of a Bacillus secretion leader sequence that
functions for secretion culturing said Bacillus subtilis under
conditions suitable for the production and secretion of said
predetermined protein, and recovering said predetermined protein as
a secreted product of said Bacillus subtilis bacteria.
10. A method according to claim 9 wherein said transport mechanism
provided for by a functional portion of the Bacillus secretion
leader sequence is a portion of the .alpha.-amylasegene.
11. A method according to claim 9 wherein said gene for said
predetermined protein is under the control of the beta-lactamase
operator, promoter, and ribosomal binding site sequence.
12. A method according to claim 9 wherein said predetermined
protein is beta-lactamase.
13. A method according to claim 9 wherein said predetermined
protein is a mammalian hormone.
14. A method according to claim 9 wherein said transport mechanism
is provided by a portion of the Bacillus licheniformis
.beta.-lactamase secretion leader that functions for secretion.
15. A method according to claim 9 wherein said predetermined
protein is a mammalian protein.
16. A hybrid cloning vector for producing a predetermined protein
through expression of B. subtilis, comprising a vector which is
capable of autonomous replication in B. subtilis and having a gene
therein for said predetermined protein, said gene being located and
oriented in said vector such as to be under the control of an
operator, promoter, and ribosomal binding site sequence, said
predetermined protein being under the control of a transport
mechanism comprising a portion of a Bacillus secretion leader
sequence that functions for secretion by which said predetermined
protein is secreted by B. subtilis.
17. A hybrid cloning vector according to claim 16 wherein said
predetermined protein is a mammalian protein.
18. A hybrid cloning vector according to claim 16 comprising a
plasmid selected from the group consisting of: pC221, pUB110,
pC194, pUB112, pT127, pOG2165, pOG2110, pOG1196 and pCS832.
19. A hybrid cloning vector according to claim 16 wherein said gene
for said predetermined protein is under the control of the
beta-lactamase operator, promoter, and ribosomal binding site
sequence.
20. A hybrid cloning vector according to claim 16 wherein said
predetermined protein is beta-lactamase.
21. A hybrid cloning vector according to claim 16 wherein said
predetermined protein is a mammalian hormone.
22. A cloning vector according to claim 16 wherein said transport
mechanism is provided for by a portion of the Bacillus
licheniformis .beta.-lactamase secretion leader that functions for
secretion.
23. A cloning vector according to claim 16 wherein said transport
mechanism provided for by a functional portion of a Bacillus
secretion leader sequence is a portion of the .alpha.-amylase
gene.
24. A hybrid cloning vector comprising a plasmid selected from the
group consisting of pOG2110, pOG1196 and pOG2165.
25. A method for creating a microorganism capable of producing a
predetermined protein through expression, comprising, forming a
hybrid cloning vector capable of autonomous replication in B.
subtilis and having a gene therein for said predetermined protein,
said predetermined protein being non-indigenous to B. subtilis,
said gene being located and oriented in said vector such as to be
under the control of an operator, promoter and ribosomal binding
site sequence, said predetermined protein being under the control
of a transport mechanism comprising a portion of a Bacillus
secretion leader sequence that functions for secretion by which
said predetermined protein is secreted by B. subtilis and
introducing said vector to B. subtilis.
26. A method according to claim 25 wherein said transport mechanism
provided for by a functional portion of a Bacillus secretion leader
sequence is a portion of the .alpha.-amylase gene.
27. A method according to claim 25 wherein said transport mechanism
is provided for by a portion of the Bacillus licheniformis
.beta.-lactamase secretion leader that functions for secretion.
28. A method according to claim 25 wherein said gene for said
predetermined protein is under the control of the beta-lactamase
operator, promoter, and ribosomal binding site sequence.
29. A method according to claim 25 wherein said protein is
beta-lactamase.
30. A microorganism capable of producing a predetermined protein
through expression, comprising a host microorganism, a hybrid
cloning vector capable of autonomous replication in said host; said
vector having a gene therein for said predetermined protein; said
gene being for a predetermined protein which is non-indigenous to
said host microorganism; said gene being located and oriented in
said vector such as to be under the control of an operator,
promoter and ribosomal binding site sequence, said predetermined
protein being under the control of a transport mechanism comprising
a portion of a Bacillus secretion leader sequence that functions
for secretion by which said predetermined protein is secreted by
said host microorganism.
31. A transformant bacterial culture cloned from a microorganism
according to claim 30, the members of said culture being capable of
expressing said predetermined protein.
32. A microorganism according to claim 30 wherein said transport
mechanism provided for by a functional protein of a Bacillus
secretion leader sequence is a portion of the .alpha.-amylase
gene.
33. A microorganism according to claim 30 wherein said host
microorganism is selected from the group consisting of B. subtilis
and E. coli.
34. A microorganism according to claim 30 wherein said hybrid
cloning vector comprises a plasmid selected from the group
consisting of pC221, pUB110, pC194, pUB112, pT127, pOG2165,
pOG2110, pCS1006, pOG1196, pCS832.
35. A microorganism according to claim 30 wherein said gene or said
predetermined protein is under the control of the beta-lactamase
operator, promoter, and ribosomal binding site sequence.
36. A microorganism according to claim 30 wherein said
predetermined protein is a mammalian protein.
37. A microorganism according to claim 30 wherein said
predetermined protein is a mammalian hormone.
38. A microorganism according to claim 30 wherein said
predetermined protein is beta-lactamase.
39. A microorganism according to claim 30 wherein said hybrid
vector is a bifunctional plasmid vector having unique sites for
restriction enzymes SstI, HindIII, PstI, and BglII.
40. A microorganism according to claim 30 wherein said transport
mechanism is provided for by a portion of the Bacillus
licheniformis .beta.-lactamase secretion leader that functions for
secretion.
Description
This invention relates to molecular biology and, more particularly,
to the so-called art of recombinant DNA. Specifically, the
invention relates to a method and a cloning vector for the
controlled accumulation of cloned heterologous gene product in
Bacillus subtilis and other bacteria, thereby facilitating the
recovery of the gene product.
The invention discloses three unique genetically engineered
plasmids. Organisms containing these plasmids have been deposited
with the American Type Culture Collection, Rockville, Md., 20852.
They have been assigned ATCC numbers: 31,776-31,778. ATCC number
31,776 has been assigned to plasmid pOG1196; number 31,777 has been
assigned to plasmid pOG2165; and number 31,778 has been assigned to
plasmid pOG2110. Applicant has directed that the plasmids be freely
available to the general public upon the issuance of a U.S.
patent.
As is well known, the particular sequence of amino acids in a given
protein is determined in accordance with the code carried in the
gene for that protein. In the process of translation by which
proteins are formed from DNA, via messenger RNA, groups of three
nucleotides in the DNA, called codons, each place one of twenty
possible amino acids at a corresponding position in the protein
chain.
With the advent of recombinant DNA techniques, genetic changes may
be made deliberately by the introduction of a predetermined
nucleotide sequence, either synthesized or isolated from one strain
or species, into the genetic makeup of another strain or species.
The known nucleotide sequence may be selected to cause the strain
or species into which it is introduced to produce, as part of the
translation process, the protein encoded by the known nucleotide
sequence. When the modified strain or species proceeds with the
normal replication process, it also then duplicates the inserted
sequence.
Recombinant DNA techniques involve isolating a suitable piece of a
DNA chain (a cloning vector) and breaking or severing the two
strands of DNA of the cloning vector at the desired location where
the foreign DNA is to be inserted. To do this, particular types of
proteins, called restriction enzymes, are typically used.
Restriction enzymes will break the DNA at particular nucleotide
sequences, although with some restriction enzymes the break may not
necessarily occur at the same point on the two intertwined DNA
strands. In such a case, if two different types of DNA are severed
in a similar manner, the open ends will be complementary and will,
under suitable conditions, stick together with the complementary
ends lying side by side. They may then be linked together
enzymatically (with ligase). This makes it possible to recombine
two DNA segments from any source into a single DNA molecule.
Once the DNA vector has been isolated and the foreign piece
inserted therein, the recombinant DNA is then placed into a
suitable host organism. In order for the host organism to replicate
the inserted DNA, it is necessary that the recombinant DNA be
inserted into the host in such a way as to become part of its
genetic system.
For example, in the bacterium Escherichia coli, two convenient
types of cloning vectors have been utilized. E. coli bacteria, in
addition to the main DNA chain or chromosome, frequently have one
or more independently replicating circular loops of DNA known as
plasmids. Also, a certain type of virus known as a lambda
bacteriophage (phage) is also capable of infecting E. coli and
becoming part of its genetic system. Recombinant DNA techniques
have included the use of a variety of plasmids or phages as cloning
vectors. This involves the isolation of plasmids or phages from the
bacteria, the breaking open of the isolated DNA by restriction
enzymes, the insertion of a foreign or heterologous piece of DNA
into the plasmid or phage, the restoration of the circular form of
the plasmid or the phage structure, and the return of the plasmid
or phage to the E. coli cell. Once in the host, the heterologous
DNA is not only replicated from generation to generation, but also
will produce the protein for which it codes if the proper reading
frame and promoters exist.
Once heterologous DNA has been successfully recombined into a host
microorganism, and the microorganism has produced the cloned gene
product, the desired product must be recovered. To do this it has,
up to the present invention, been necessary to destroy the cells
producing the desired product in order to harvest the product
itself. Also, because cells naturally contain a great many
different proteins, the isolation process for the desired product
may be difficult or complex. Finally, the desired product may be
detrimental to the host cell, particularly if it is produced at a
high level. In some cases, this may result in destruction of the
cells, and in other cases it may result in the cells activating a
defensive mechanism to degrade the desired product.
Most recombinant DNA work to date has been carried out with E.
coli. E. coli is a member of the Gram negative class of bacteria
which contain two layers of membranes enclosing a periplasmic
space. Many of the products produced in E. coli are secreted into
this periplasmic space, if secreted at all. Few products are
secreted outside the living cells into the growth media.
On the other hand, Bacillus subtilis is a member of the gram
positive class of bacteria which contain only a single layer of
bacterial membrane. Thus B. subtilis can produce large amounts of
protein which are secreted directly into the growth media. Although
the general approach to gene cloning in E. coli is applicable to B.
subtilis, attempts to produce a useful product of heterologous gene
cloned into B. subtilis and secreted into the growth media have
thus far been unsuccessful. B. subtilis is somewhat preferable to
E. coli because of a greater efficiency for plasmid mediated
transformation and because it is non-pathogenic.
It is an object of the present invention to provide an improved
method for producing heterologous protein by microorganisms.
Another object of the invention is to provide a method for
producing a predetermined protein through expression by B.
subtilis, such protein being non-indigenous to B. subtilis.
A further object of the invention is to provide a method for
producing protein by B. subtilis wherein the protein is
non-indigenous to the host species and is secreted into the growth
media.
Another object of the invention is to provide a method for
producing protein by E. coli wherein the protein is non-indigenous
to the host species and is secreted into the periplasmic space.
A further object of the invention is to provide a plasmid for
producing a predetermined protein through expression by B. subtilis
or E. coli, such protein being non-indigenous to B. subtilis.
Other objects of the invention will become apparent to those
skilled in the art from the following description, taken in
connection with the accompanying drawings wherein:
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic drawing providing a partial structural map of
a DNA fragment which contains the gene for beta-lactamase obtained
from Bacillus licheniformis;
FIG. 2 is a schematic drawing indicating correspondence between a
number of the nucleotide sequences in the fragment of FIG. 1 and
protein sequence present in the protein for which the fragment
codes, and also indicating the corresponding restriction enzyme
recognition sites;
FIG. 3 is a drawing indicating the typical appearance of plates
upon which Bacillus subtilis strains harboring the recombinant
plasmids are grown, as the plates appear after PVA assay for
beta-lactamase activity;
FIG. 4 is a schematic illustration of the construction of E. coli
plasmid pOG2110, B. subtilis plasmid pOG1196, and the bifunctional
plasmid pOG2165;
FIG. 5 is a drawing indicating the typical appearance of plates
upon which various bacterial strains harboring the recombinant
plasmids are grown, as the plates appear after PVA assay for
beta-lactamase activity; and
FIG. 6 is a diagram of the nucleotides comprising a portion of the
B. licheniformis Pen P gene.
DETAILED DESCRIPTION OF THE INVENTION
Very generally, and in accordance with one form of the invention, a
predetermined protein which is non-indigenous to B. subtilis is
produced through expression by B. subtilis. Growth media and
conditions are provided for growing a strain of B. subtilis in
which a plasmid has been introduced. The plasmid is capable of
being replicated in the strain and has a gene therein for the
predetermined protein. The gene is located and oriented in the
plasmid such as to be under the control of an operator, promoter
and ribosomal binding site sequence. The protein is also under the
control of a transport mechanism by which the protein is secreted
by the host strain. Upon secretion, the protein is recovered from
the growth media.
When, according to the invention, the predetermined protein is
expressed in a Gram negative bacteria, such as E. coli, the protein
is again transported across the bacterial membrane by the transport
peptide. However, here the protein is "secreted" into the
periplasmic space instead of the growth media since the Gram
negative bacterium has a cell wall in addition to its normal
bacterial membrane. The predetermined protein can be recovered from
the periplasmic space.
The method of the invention necessitates the use of a cloning
vector organism containing a sequence of nucleotides capable of
initiating the transcription and translation process. These
nucleotides, which provide an operator, promoter, and ribosomal
binding site sequence, may be naturally present in the vector, may
be inserted therein as a separate segment of DNA using recombinant
DNA techniques, or may be part of the heterologous DNA containing
the gene of interest. The heterologous DNA, which will contain at
least the structural gene for a desired protein product, is placed
in the cloning vector so as to be transcribed and translated under
control of the operator, promoter, and ribosomal binding site
sequence. For correct translation of the inserted heterologous DNA,
the nucleotides in the inserted DNA must be in the correct reading
frame. In addition, it may be desirable or even necessary that the
cloning vector include a sufficient number of nucleotides
indigenous to the host cell to ensure read-through translation from
the operator, promoter, and ribosomal binding site sequence into
and through the inserted heterologous DNA in the correct reading
frame.
In accordance with the invention, the cloning vector utilized
includes a sequence of nucleotides which comprise codons for a
functional transport signal peptide sequence. Transport signal
peptide sequences are typically short leader sequences of amino
acids on newly made proteins. Although the mechanism by which the
transport signal peptide sequence operates is not entirely
understood, it is believed that the transport protein is excreted
by the cell and withdraws that protein appended to it from the
cytoplasm as the protein is made. Once the transport function has
been performed by the transport signal peptide sequence, the
transport sequence may be removed by natural processes.
In accordance with the invention, the heterologous DNA can be
inserted into the cloning vector at a location which allows the
protein for which it encodes to be transported in accordance with
the transport signal peptide sequence coded by the signal codons.
Thus, the cloned gene product can be conveniently transported to a
desired destination from which the gene product may be harvested.
This has several advantages. Because the destination is outside the
cell, the host cells need not be destroyed to harvest the gene
products, thus allowing for the continuous or uninterrupted
production of the gene product. Also, since cells contain a great
many proteins, the ability to export the cloned gene product makes
the isolation and purification of the product a much simpler task.
Finally, since cloned gene products, especially if produced at a
high level, may be detrimental to the host cells, the ability to
harvest the cloned gene products from outside of the cell membranes
often means that the products will not harm the cells, nor will the
cells possibly produce a defensive enzyme which will degrade the
gene product.
The precise location at which the heterologous DNA must be inserted
in the cloning vector will, of course, depend upon how the
transport signal peptide sequence functions. In some cases, the
transport signal peptide sequence will immediately precede the
heterologous DNA, either as part of the gene itself or having
already been present in the plasmid. The signal sequence itself can
constitute the necessary sequence of nucleotides to provide for
readthrough translation of the heterologous DNA. On the other hand,
there may be some cases in which the transport signal peptide
sequence must be located elsewhere than immediately preceding the
heterologous DNA. In such cases, it may be necessary to produce the
desired peptide sequence with some additional amino-acids at the
beginning (coded by the extraneous codons) in order to provide the
necessary readthrough functions.
The following examples illustrate other specific instances in which
the invention may be employed, but are not intended to limit the
scope of the invention:
EXAMPLE 1
In order to provide a plasmid for producing a predetermined protein
non-indigenous to a host organism, a plasmid vector containing the
B. licheniformis beta-lactamase gene was made and then replicated
in both B. subtilis and E. coli. The plasmid was constructed by
purifying a 3.5 kb (kilobase) EcoRI-SstI fragment containing the
beta-lactamase gene and then ligating it with a 2.1 kb. EcoRI-SstI
fragment containing the replication function of the E. coli plasmid
pOP1.DELTA.6 (described by Gelfand, et al., Proc. the Nat. Acad.
Sci. USA (1978) 75, 5869-5873). Following transformation into
competent E. coli CS412 cells (an r.sub.k.sup.- m.sub.k.sup.+
Pro.sup.- derivative of C600) and a growth period adequate for
expression (90 minutes), Ap-resistant transformants were obtained.
Plasmids from three clones were prepared; one designated as pOG2110
was further characterized. FIG. 4 details the location of the
expected and observed restriction sites in pOG2110.
To allow for replication of pOG2110 in B. subtilis, a bifunctional
replicon was constructed using pOG2110 and the B. subtilis plasmid
pOG1196. The construction of pOG1196 is summarized in FIG. 4.
Initially a chimeric plasmid (pCS832) containing the entire
sequences of plasmids pC194 (Cm.sup.R) and pUB110 (km.sup.R) was
made by ligating the two MboI fragments of pC194 with the BamHI
digested pUB110. The resulting plasmid carried both the Cm gene
from PC194 and the Km (Nm) gene from pUB110. It has a size of 7.5
kb. A spontaneous deletion mutant (plasmid pCS1006) was obtained
from one of the sub-clones. It had lost the HpaII site originated
in pC194 and known to be located in the pC194 replication region
(Chang and Cohen, Molec. Gen. Genet., (1979) 168, 111-115). It
still retained the replication function of pUB110 and the two
resistance markers. By recirculating the largest HpaII fragment
(3.6 kb) of PCS1006, plasmid pOG1196 was obtained. This plasmid
confers only Cm-resistance and possesses the replication function
derived from plasmid pUB110. The map of pOG1196 is shown in FIG.
4.
E. coli plasmid pOG2110 and B. subtilis plasmid pOG1196 contained
two and three PvuII sites respectively. Equal amounts of PvuII
digested pOG2110 and pOG1196 plasmid DNA were ligated and used to
transform E. coli strain CS412. Cm-resistant clones were selected;
all were also Ap-resistant. The composite plasmid pOG2165 isolated
from one of the Cm-resistant Ap-resistant transformants was studied
further. A map of this 7.5 kb plasmid is shown in FIG. 4. Plasmid
pOG2165 replicates in both E. coli and B. subtilis and confers upon
either host both Cm- and Ap-resistance.
B. subtilis and E. coli cells harboring plasmid pOG2165 are
resistant to ampicillin as a result of the production of the B.
licheniformis beta-lactamase enzyme. This can be demonstrated by
means of the PVA plate assay developed by Sherratt and Colling
(Ref. 9). The positive results obtained from such an assay are
illustrated in FIG. 5.
When pOG2165 is propagated in B. subtilis BD224, both the
membrane-bound and the secreted form of the heterologous
beta-lactamase are synthesized. The amount produced by this strain
is variable and depends upon the growth conditions used. pOG2165
can also be propagated in B. subtilis strain QB127 (Kunst et al,
Bio. Chemie. (1974) 56, 1481-1490). QB127 is a bacterial strain
with the sacU.sup.h mutation which causes overproduction of several
exoenzymes such as levan-sucrase, alpha-amylase and extra-cellular
proteases. Levels of beta-lactamase detected in cultures of QB127
(pOG2165) are similar to those levels detected in BD224 (pOG2165)
cultures under the same conditions.
The bi-functional plasmid pOG2165 itself possesses unique sites for
restriction enzymes SstI, HindIII, PstI and BglII. Insertion of DNA
into the BglII and the PstI sites leads to inactivation of the B.
licheniformis beta-lactamase gene and provides an easily
recognizable phenotype for identifying clones carrying inserts.
When the exact reading frame of the DNA sequence to be inserted is
known, it is possible to create a fused protein containing the
leader sequence and the first 71 amino acid residues of the
beta-lactamase exoenzyme by cloning into the BglII site. Fused
protein made in this way is secreted by the Bacillus cells due to
the presence of the leader sequence at the amino terminus. These
features make pOG2165 a useful vector for the cloning and efficient
expression of heterlogous genes, and the subsequent secretion of
the gene product in B. subtilis and E. coli.
On the other hand, when the exact reading frame of the DNA sequence
to be inserted is known, and insertion is made at the PstI site, a
fused protein is made which will accumulate in the host organism.
Since the PstI recognition site is located in the initial portion
of the nucleotide sequence coding for the signal peptide, only a
portion of this sequence is transcribed before the heterologous
gene sequence is encountered. Even though the fused protein is
expressed, this portion of the signal peptide is insufficient to
confer the normal signal peptide's secretion function. As a result,
products of genes inserted at the PstI site accumulate in the host
organism.
A portion of the nucleotide sequence comprising the B.
licheniformis Pen P gene is diagrammed in FIG. 6. The
beta-lactamase promoter region is located between nucleotides 1 and
221 one; its precise location is unknown. The nucleotides which
code for the amino acids comprising the signal peptide begin with
nucleotide 222 and end with nucleotide 323. As a result, the signal
peptide is composed of thirty-four amino acids. The PstI
recognition site is located between nucleotide positions 259 and
264, or at amino acids thirteen through fifteen on the signal
peptide chain. Insertion of heterologous DNA into this PstI site
will lead to the formation of a fused protein composed of the
heterologous DNA product and the first fourteen amino acids of the
signal peptide chain. Such a fused protein will be expressed but
not transported across the bacterial membrane. Successful secretion
requires fusion with either the total signal peptide, or fusion
with at least the first twenty six amino acid residues in the
signal peptide chain.
EXAMPLE 2
B. licheniformis produces a large amount of beta-lactamase in the
secreted form (exo-enzyme). The secretion of this protein is
believed to be the result of the interaction between the bacterial
membrane and the amino acid leader sequence which facilitates the
transport of the protein across the single bacterial membrane
barrier. The beta-lactamase gene was cloned and inserted into
plasmids which were capable of being replicated in B. subtilis. The
plasmids were then transformed into the B. subtilis hosts resulting
in the secretion of beta-lactamase. This constitutes the first
expression of a heterologous gene in B. subtilis and the transport
of the gene products into the culture or growth media from the B.
subtilis cells.
To clone the beta-lactamase gene from the B. licheniformis strain,
total chromosomal DNA from B. licheniformis strain 749/C was
isolated and digested with EcoRI restriction endonuclease.
Chromosomal DNA isolated from B. licheniformis 749/C was prepared
according to Marmur (J. of Molec. Biol. (1961) 3, 208-18). E. coli
plasmid pSC101 is isolated from cells using the cleared lysate
procedure of Kupersztoch and Helinski (Biochem. Biophys. Res.
Commun. (1973) 54, 1451-59). 3 .mu.g of chromosomal DNA and 2 .mu.g
of pSC101 DNA were digested with endonuclease EcoRI and ligated
with T4 DNA ligase as described (Hershfield, et al. Proc. Natl.
Acad. Sci., USA (1974) 71, 3455-59), and transformed into competent
cells of E. coli strain CS412 (an r.sub.k.sup.- m.sub.k.sup.+
Pro.sup.- derivative of C600) using the protocol of Cohen, et al.
(Proc. Natl. Acad. Sci., USA (1972 ) 69, 2110-14).
Transformants resistant to ampicillin at 10 .mu.g/ml were selected
and one of the transformants carrying recombinant plasmid
designated pTB2 was characterized further. Plasmid pTB2 carries a
4.2 kb (kilobase pair) EcoRI fragment on the pSC101 vector. This
plasmid confers to the host tetracycline (the marker on pSC101) and
ampicillin resistances, indicating that the beta-lactamase gene
product is made as a functional enzyme which degrades ampicillin in
the media.
The beta-lactamase gene is located on the 4.2 kilobase pair EcoRI
fragment mentioned above. Subsequent analysis of this fragment
using various restriction enzymes and gene cloning permits
deduction of the structure of the gene for this enzyme is partially
mapped and shown in FIG. 1. The primary sequence of the
beta-lactamase from B. licheniformis strain 749/C has been
previously determined (R. J. Meadway, Ph. D., Thesis, University of
Edinburgh, 1969). From the known amino acid sequence, the Gly-Pro
(position 116-117) sequence corresponds to the nucleotide sequence
GGN-CCN, which in turn is the recognition sequence for endonuclease
Sau96I (GGNCC). Similarly, the Trp-Pro (position 222-223) sequence
is coded by nucleotide codons TGG-CCN, within which the center
tetranucleotide sequence GGCC is recognized and cleaved by
endonuclease HaeIII (see Roberts, in DNA Insertion Elements,
Plasmids, and Episomes, 1977, ed. Bukhari, Shapiro and Adhya, Cold
Spring Harbor Lab., p. 757).
The 4.2 kb cloned fragment was analyzed by a number of
endonucleases, as listed in FIG. 2, using the conditions specified
by the supplier (New England Biolabs, Inc., Beverly, MA 01915, 1978
catalog). The digested DNA was analyzed on agarose gels as
described by Sharp, et al. (Biochemistry (1973) 12, 3055-63), and
on acrylamide gels (Maxam and Gilbert, Proc. Natl. Acad. Sci., USA
(1973) 73, 3942-46). The mapping data is summarized in FIG. 2. The
Sau961 site and the HaeIII site were located in the 2.3 kb PvuII
fragment which contains the complete beta-lactamase gene sequence.
These two sites are separated by 320 nucleotides which is
consistent with the protein sequence data (106 amino acids
apart).
After identification of the nucleotide sequence which contains the
beta-lactamase gene, the gene was cloned into B. subtilis using
various Bacillus plasmids and using a hybrid B. subtilis-E. coli
plasmid. Plasmids included pUB110 and pC221 derived plasmids. The
B. subtilis strains harboring the recombinant plasmids become
resistant to ampicillin as well as give a positive beta-lactamase
reaction on PVA plates (see FIG. 3). Furthermore, beta-lactamase
activity was detected in cultures after bacterial cells were
removed. This activity clearly indicates the successful expression
of the heterologous gene in B. subtilis, as well as the transport
of the protein through the bacterial membrane into the culture or
growth media.
The EcoRI fragment containing the beta-lactamase gene was cloned
onto B. subtilis plasmid vectors pUB110 (Ref. 5) and pC221 (Erlich,
Proc. Natl. Acad. Sci., USA (1977) 74, 1680-82) at the respective
EcoRI sites using the procedure described above in connection with
E. coli. Similarly, the 2.3 kb PvuII fragment containing the
beta-lactamase gene has also been cloned onto pUB110 at the PvuII
site and the TacI site.
Ligated DNA preparations were used to transform B. subtilis strain
BD224 (recE4, trpC2, thr5) by the method of Chang and Cohen (Ref.
7). Transformants resistant to ampicillin on regeneration plates
were selected and tested. The production of beta-lactamase is
detected by two methods; one is a sensitive plate assay developed
by Sherratt and Collins (Ref. 9), the other is the iodometric assay
described by Ross and O'Callaghan (Meth. in Enzymology, (1975) 43,
69). B. subtilis clones resistant to ampicillin gave positive
results for both tests. In addition, beta-lactamase activity was
also detected in the culture after the cells were removed,
indicating that the beta-lactamase is not only made, but also
exported in B. subtilis.
The cloning of heterologous genes into B. subtilis and the
functional expression of these genes as intracellular proteins have
been shown previously by Keggins et al., Ref. 6. In this study,
however, the cloned genes are genes coding for enzymes which are
normally present intracellularly in wild type B. subtilis. The
successful expression of genes which are non-indigenous to B.
subtilis, and the successful secretion of the products of these
genes was not demonstrated.
The work on the beta-lactamase gene presented in this Example is
the first demonstration that a new function namely, beta-lactamase
production, can be introduced into B. subtilis using gene cloning
techniques. In addition, this Example constitutes the first
demonstration that a foreign gene product can be made to pass
through the B. subtilis membrane barrier and be secreted as an
exo-protein. The beta-lactamase, which is a commercially useful
product, is produced by a strain which does not otherwise produce
this enzyme.
EXAMPLE 3
The B. licheniformis beta-lactamase gene described in Example 2 was
also inserted into plasmids capable of being replicated in E. coli.
These plasmids were transformed into the E. coli hosts using
methods identical to those described in Example 2 for B. subtilis.
The E. coli cells harboring the plasmids are resistant to
ampicillin as a result of the production of the B. licheniformis
beta-lactamase enzyme. This is demonstrated by means of the PVA
plate assay developed by Sherratt and Collins (Ref. 9).
When the B. licheniformis beta-lactamase gene carrying plasmids are
propagated in E. coli, the secreted form of the enzyme is not
transported into the culture medium as it is in B. subtilis. Since
E. coli has a cell wall surrounding the limiting bacterial
membrane, the heterologous protein product is transported to the
periplasmic space separating the cell wall from the bacterial
membrane. The beta-lactamase exoenzyme was allowed to accummulate
in the periplasmic space and was then harvested by appropriate
methods.
EXAMPLE 4
The beta-lactamase gene of B. licheniformis is not the only source
of a signal peptide sequence that can function in B. subtilis or E.
coli. As indicated above, many proteins (especially in eucaryotic
cells) are transported across membranes. Although the precise amino
acid sequence of the "signal region" may, and in fact does, vary
among different transported proteins, the folded structures of
these regions, which can be predicted according to the rules of
Chou and Fasman (Ann. Rev. Biochem. (1978) 47, 251), are very
similar. Thus, the requirements for a transport signal peptide
sequence can be satisfied with non-Bacillus signal peptides,
provided the signal sequence encoding DNA fragment is correctly
positioned downstream from a Bacillus promoter, and ribosomal
binding site sequence.
One such eucaryotic transport signal sequence that may be used in
connection with a desired gene product is the signal or presequence
preceding the insulin B chain. (The various insulin chains, A, B
and C, are made as a single polypeptide and are assembled and
processed in the endoplasmic reticulum on the way to being exported
by the cell). However, in the insulin presequence there is no
convenient restriction site immediately after the signal peptide
sequence which can be used to join it with a cloned gene of
heterologous DNA. Nevertheless, the last five nucleotides of the
signal sequence in insulin are AGGCT and the first nucleotide of
the insulin B chain itself is T. These six nucleotides together
(AGGCTT) differ by a single nucleotide from AAGCTT which is the
recognition sequence for the restriction enzyme HindIII. The enzyme
cuts between the two As. If the heterologous DNA was cloned or
separated using the same enzyme, HindIII, or using a half HindIII
site bifunctional linker, the sequence will be restored when the
heterologous DNA is inserted.
The single nucleotide G.sub.x may be changed to an A to provide the
HindIII site by the procedure described by Bahl in U.S. patent
application Ser. No. 128,671 or by the procedure described by Bahl
in U.S. patent application Ser. No. 133,150, U.S. Pat. No.
4,351,901, issued Sept. 28, 1982. In the first case, a small single
stranded fragment incorporating the change is synthesized by the
method of Narang et al. (Canadian Journ. of Chem. 51:3649). In the
second procedure, the nucleotide to be changed is exposed, altered,
and the sequence reconstructed. In either case, this change results
in no alteration of the next to last amino acid of the signal
sequence.
DNA ligation may be accomplished as described by Hershfield et al.
(Proc. Natl. Acad. Sci. USA 71:3455). Reverse transcriptase may be
used as a DNA polymerase such as described by Bahl et al. (Proc.
Natl. Acad. Sci. USA 74:966). Transformation may proceed as
described by Cohen et al. (Proc. Natl. Acad. Sci. USA 70:3240).
EXAMPLE 5
Although the procedure described in Example 4 above is technically
feasible, it is a eukaryotic leader sequence and thus the procedure
may be useful only in connection with general research. Typically,
a more useful approach from a commercial standpoint is in
connection with a prokaryotic (bacterial) host. Although only a few
such signal sequences are known in connection with prokaryotic
systems, one sequence which is well characterized is the sequence
from TEM beta-lactamase. (Sutcliffe Proc. Natl. Acad. Sci. USA
75:3737). This sequence is ideal for attaching to a cloned gene
except that there is no convenient restriction site at the location
for presequence processing. The nearest restriction site to the
signal sequence is an MboI site which would result in the
attachment of 16 extraneous amino acids to the cloned gene
product.
Nevertheless, the terminal portion of the signal sequence, which is
TTTGCT, may be altered, by one of the above-described techniques,
to TTTGAT. When the latter sequence is read along with the first
few following nucleotides of the TEM beta-lactamase gene, a
restriction site for BclI (TGATCA) exists. This results in the
altering of the final amino acid from Ala to Asp and in the
attaching of one extra amino acid to the gene product, either a Glu
or His, depending upon what the first attached nucleotide of the
heterologous DNA is.
To accomplish the foregoing alteration in nucleotides, the approach
described above may be used wherein a short fragment is
synthesized. There are bracketing restriction sites upstream from
the signal sequence (ThaI) and a restriction site MboI, as
mentioned above, downstream. A Taq site exists even further
downstream. Restriction conditions may be followed for ThaI as
described by McConnell et al., Nucleic Acid Res. 5:1729; for MboI
by Galinas et al., J. Mol. Biol. 114:169; or TaqI by Sato et al.,
Proc. Natl. Acad. Sci. USA 74:542. Note that in connection with
MboI, the DNA is prepared in the host cell GM119 which lacks
deoxyadenosine methylase (to avoid methylation of regions and
prevent MboI cutting). In this latter connection see Marinus et
al., Mutat. Res. 28:15. Alternatively, the restriction endonuclease
SAU 3A, an isoschizome of MboI, may be used with DNA isolated from
any host.
It may be seen, therefore, that the invention provides a method and
a vector for the controlled accumulation of heterologous cloned
gene products. The products are transferred or transported outside
the host cell, enabling the harvesting of the product to proceed
with minimal restrictions, and avoiding the likelihood of
degradation or destruction of either the host cell or the gene
product itself.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and accompanying drawings. Such
modifications are intended to fall within the scope of the appended
claims.
REFERENCES
1. Cohen, S. N., Chang. A. C. Y., Boyer, H. W., and Helling, R. B.,
(1973) Proc. Natl. Acad. Sci. USA 70, 3240-4.
2. Collins, J., (1977) Curr. Top. Microbiol. Immunol. 78,
121-170.
3. O'Farrell, P. H., Polisky, B., and Gelfand, D. H., (1978) J.
Bacteriol. 134, 645-654.
4. Polisky, B., Bishop, R. J., Gelfand, D. H., (1976) Proc. Natl.
Acad. Sci. USA 73, 3900-3904.
5. Gryczan, T. J., and Dubnau, D., (1978) Proc. Natl. Acad. Sci.,
USA 75, 1428-1432.
6. Keggins, K. H., Lovett, P. S., and Duvall, E. J., (1978) ibid
1423-1427.
7. Chang, S., and Cohen, S. N., (1979) Molec. Gen. Genet. 168,
111-115.
8. Chang, A. C. Y., and Cohen, S. N., (1974) Proc. Natl. Acad. Sci.
USA 71, 1030-1034.
9. Sherratt, D. J., and Collins, J. F., (1973) J. Gen. Microbiol.
76, 217-230.
* * * * *