United States Patent
`Uhlin et al.
`
`[19]
`
`[11] Patent Number:
`
`4,487,835
`
`[45] Date of Patent:
`
`Dec. 11, 1984
`
`[54] PLASMID HAVING TEMPERATURE
`DEPENDENT PLASMID COPY NUMBER
`Bernt E. Uhlin; Kurt Nordstrfim;
`Inventors:
`Soeren Molin, Petter Gustaffson,
`all of Odense Denmark
`
`[75]
`
`[73] Assignee:
`
`A/S Alfred Benzon, Copenhagen,
`Denmark
`
`[21] App1.No.: 214,423
`[22] Filed:
`Dec. 8, 1980
`
`Related U.S. Application Data
`Division of Ser. No. 908,108, May 22, 1978.
`
`[62]
`
`Foreign Application Priority Data
`[30]
`Dec. 30, 1977 [DE]
`Fed. Rep. of Germany ....... 2759053
`
`Int. c1.3 ........................ C12N1/00;C12N15/O0
`[51]
`[52] U.S.C1. .................................... .. 435/317; 935/29;
`935/43; 435/172.3
`[58] Field of Search ........................ ..43s/317,172,618
`
`[5 6]
`
`References Cited
`PUBLICATIONS
`
`Goebel, Eur. J. Biochem., 43, pp. 126-130, (1974).
`Journal of Bacteriology, Jan. 1975, pp. 354-362.
`“Construction of a Colicin E1-R Factor Composite
`Plasmid in Vitro Means for Amplification of Deoxyri—
`bonucleic Acid”, by Tanaka et al., Journal of Bacteriol-
`ogy, May l972, pp. 529-537.
`“Properties of an R Factor Which Originated in Pseudo-
`monas aeruginasa 1822", by John Grinsted et al.
`Genet. Res., Camb. (1968), 12, pp. 169-185, “Resistance
`of Escherichia calf to penicillins”, by Hans G, Boman et
`al.
`Genetics 39, Jul. 1954, “Segregation of New Lysogenic
`Types During Growth of a Doubly Lysogenic Strain
`Derived from Escltericltia call", by R. K. Appleyard.
`Journal of Bacteriology, Apr. 1976, pp. 1-6, “Translo—
`cation of a Discrete Piece of Deoxyribonucleic Acid
`Carrying an amp Gene Between Replicons in Escher-
`ichia coli”, by P. M. Bennett et al.
`Nature, vol. 214, May 27, 1967, “Mutant Drug Resistant
`
`Factors of High Transmissibility”, by Elinor Meynell et
`al.
`Molec., gen. Genet., 142, 239-249, (1975), “The Gener-
`ation of a ColE1-Ap Cloning Vehicle which Allows
`Detection of Inserted DNA”, by Magdalene So et al.
`Journal of Bacteriology, May 1972, (pp. 562-569), “Mu-
`tations in R Factors of Escherichia call‘ Causing an In-
`creased Number of R-Factor Copies per Chromo-
`some”, by Kurt Nordstrom et al.
`Journal of Bacteriology, Aug. 1975, pp. 658-665, “Class
`of Small Multicopy Plasmids Originating From the
`Mutant Antibiotic Resistance Factor Rldrd-l9B2”, by
`Werner Goebel et al.
`Cell, vol. 11, 11-23, May 1977, “Translocatable Ele-
`ments in Procaryotes”, Review by Nancy Kleckner.
`Abstract entitled, “A Mutant Plasmid with Altered
`Replication Control”, by Grindley et 3.1., Distributed at
`Second ASM Meeting on Extrachromosomal Elements,
`Jackson, Wyoming, 2/20-25/77, No. 5.
`Microbiology—1978, Edited by David Schlesinger,
`1978, pp. 71-73.
`Abstract Distributed at Workshop on Plasmids and
`Genetic E Change between Bacteria, Berlin, May
`22-25, 1976.
`Abstract entitled, “Control of Plasmid R1 Replication
`in Esc/zerichia coli: Genetics, Selection, Timing and
`Control Parameters", by Nordstrom et al., distributed a
`Second ASM Meeting on Extrachromosomal Elements,
`Jackson, Wyoming, 2/20-25, 1977, No. 1.
`(List continued on next page.)
`
`_
`Primary Examiner——Alvin E. Tanenholtz
`Attorney, Agent, or Firm—l-Iubbell, Cohen, Stiefel &
`Gross
`
`ABSTRACT
`[57]
`Gene products of plasmid DNA, such as proteins, are
`prepared in high yields by cultivating bacteria carrying
`a plasmid which shows a controlled constant plasmid
`copy number at one temperature and a much higher or
`totally uncontrolled copy number at a different temper-
`ature. The plasmid may be prepared by recombinant
`DNA technique using a cloning vector showing the
`temperature dependent plasmid copy number pattern.
`
`9 Claims, 4 Drawing Figures
`
`Sanofi/Regeneron Ex. 1033, pg 938
`
`Mylan Ex. 1033, pg 938
`
`

`
`4,487,835
`Page 2 F.
`
`OTHER PUBLICATIONS
`
`Topics in Infectious Diseases, vol. 2, R—Factors: Their
`Properties and Possible Control, “Copy Mutants of the
`Plasmid R] as a Tool in Studies of Control of Plasmid
`Replication", Nordstrom et al., pp. 299-332.
`Excerpts from Petter Gustafsson"s doctoral thesis, May
`27, 1977, pp. 29, 31, 8 and 9.
`Abstract distributed at FEMS Meeting in Paris, Dec.
`1-2, 1976, entitled “Replication Control: Copy Mutants
`of Plasmids”, Nordstrom et al.
`Abstract distributed at Symposium about R factors in
`Tokyo, Japan, Oct. 26-28, 1977, entitled “Replication
`and Copy Number Control of The R Plasmid R1 in E.
`coll”, Uhlin et al.
`Journal of Bacteriology, “Cold-sensitive Mutations in
`Salmonella typhimurium Which Affect Ribosome Syn-
`thesis”, by Tai et a1., Mar. 1969, pp. 1298-1304.
`Journal afBac1eriolog_]a “Characteristics of Cold—Sensi—
`tive Mutants of Escherichia colt’ K-12 Defective in De-
`oxyribonucleic Acid Replication”, by Wehr et al., Jul.
`1974, pp. 99-107.
`Journal ofBacteriology, Mutants of Escherichia coli with
`Cold—Sensitive Deoxyribonucleic Acid Synthesis, by
`Waskell et al., Jun. 1974, pp. 1027-1040.
`Temperature and Life, “II Genetic Regulation of Tem-
`perature Responses”, Ingraham et al., 1973, pp. 60-85.
`Proc. Soc. Gen. Microbiol., vol. 2, 1975, K. Nord-
`stroem et a1.: “Replication of the Plasmid R1”.
`
`Microbiology 1978; K. Nordstroem et a1.: “Control of
`Replication of Plasmid R1 is Escherichia colt”.
`Chemical Abstracts, vol. 88, 1978, Ref.
`l33092v, No.
`19; N. D. F. Grindley et a1.: “Mutant Plasmid with
`Altered Replication Control”.
`Chemical Abstracts, vol. 89, 6/11/1978, Ref. 159 981e,
`P. M. Andreoli et a1.: “Isolation and Characterization of
`a C10 DF1 3xTn901 Plasmid Mutant with Thermosensi-
`tive Control of DNA Replication”.
`Chemical Abstracts, vol. 83, No. 17, 27/10/1975, Ref.
`144 3935; W. Goebel et al. “Class of Small Multicopy
`Plasmids Originating From the Mutant Antibiotic Re-
`sistance Factor R1drd—19B2”.
`Chemical Abstracts, vol. 84, No. 25, 21/6/1976, ref. 176
`581p; H. Hashimoto et al.: “Isolation of a Miniplasmid
`From an R Factor“.
`Journal of Bacteriology, vol. 131, No. 2, Aug. 1977, T.
`Hashimoto et a1.: “Mutations to Temperature Sensitiv-
`ity in R Plasmid p SC 101”.
`Molec. Gen. Genet. 165, 1978; B. E. Uhlin et al.: “A
`Runaway—Replication Mutant of Plasmid Rldrd-19;
`Temperature Dependent Loss of Copy Number Con-
`trol”.
`Plasmid, 1, 1977, B. E. Uhlin and K. Nordstroem: “R
`Plasmid Gene Dosage Effects in Escherichia cali K-12:
`Copy Mutants of the R Plasmid R1drd—19”.
`Plasmid, 1, 1977, K. Nordstroem et a1.: “Phenotypic and
`Genetic Symbols for Plasmid Copy Number Mutants”.
`
`Sanofi/Regeneron Ex. 1033, pg 939
`
`Mylan Ex. 1033, pg 939
`
`

`
`US. Patent Dec. 11,1934
`
`Sheet 1 of 4
`
`4,487,835
`
`Fig.1.
`
`200
`
`100
`
`O
`
`200
`
`100
`
`AmountofDNA ("/o)
`
`
`
`Growthrate
`
`
`
`(Doublingsperhour)
`
`30
`
`32
`
`31.
`
`36
`
`Temperature (°C)
`
`Sanofi/Regeneron Ex. 1033, pg 940
`
`Mylan Ex. 1033, pg 940
`
`

`
`U151 Patent Dec. 11,1934
`
`Sheet 2 of4
`
`4,487,835
`
`Fig. 2.
`
`shift = 30°C ~——>z.0°c
`
`A
`
`A
`
`A
`v
`
`A
`
`1
`
`A
`
`=
`4 0
`
`p
`
`1
`
`.
`
`-”
`
`A
`
`'
`
`O .
`
`.
`
`.
`
`60
`
`120
`
`180
`
`21.0
`
`1000
`z-\
`2
`8
`L0
`5
`‘S
`-5
`100 L
`(-U‘
`E
`3
`3‘
`’__
`
`9.
`c:
`'6
`‘é
`‘L
`
`10
`
`1
`
`C
`E
`\1000
`t4
`E
`>,
`1..
`L‘
`‘.5
`:1
`I 100
`Q
`‘E
`2
`g
`L)
`
`)3
`
`10
`
`I
`“A
`
`./
`
`1
`0
`
`tv
`Q
`E
`.0
`‘mi
`
`TQ
`
`.
`
`Time after temperature shift (min.)
`
`Sanofi/Regeneron Ex. 1033, pg 941
`
`Mylan Ex. 1033, pg 941
`
`

`
`U..Sm Patent Dec. 11, 1984
`
`Sheet 3 0:4
`
`4,487,835
`
`Hg. 3.
`
`MO‘
`
`chromosomal DNA
`
`chromosomal DNA
`
`10
`
`‘
`
`30
`_
`20
`Fraction number
`
`40
`
`Sanofi/Regeneron Ex. 1033, pg 942
`
`Mylan Ex. 1033, pg 942
`
`

`
`US“ Patent Dec. 11,1984
`
`Sheet 4 of4
`
`4,487,835
`
`HIl|||||
`
`
`
`LII-0IG7‘nlT]UC‘)I>
`
`Sanofi/Regeneron Ex. 1033, pg 943
`
`Mylan Ex. 1033, pg 943
`
`

`
`1
`
`PLASMID HAVING TEMPERATURE
`DEPENDENT PLASMID COPY NUMBER
`
`4,487,835
`
`This is a division of application Ser. No. 903,108, filed
`May 22, 1978.
`FIELD OF THE INVENTION
`
`The present invention provides a process for produc-
`ing a gene product of plasmid DNA,
`to bacteria and
`plasmids useful
`in the process,
`to a cloning vector
`which can be used in constructing recombinant DNA
`plasmids useful in the process, and to a method of pre-
`paring such bacteria, plasmids, and cloning vectors.
`BACKGROUND OF THE INVENTION
`
`It is known to prepare useful polypeptides and prote-
`ins, for example enzymes, hormones, and (for use in e. g.
`vaccine preparation) toxins and other antigens, by culti-
`vation of bacteria carrying plasmids with genes coding
`for the desired polypeptides or proteins.
`It
`is also
`known to construct plasmids containing ‘desired genes
`by so-called recombinant DNA technique, which
`makes it possible to obtain, from the cultivated bacteria
`carrying such recombinant DNA plasmids, gene prod-
`ucts which inherently are characteristic to other organ-
`isms than the bacteria used as host cells. In the prepara-
`tion of recombinant DNA, a so-called cloning vector,
`that is, a plasmid which is able to replicate in the host
`bacterium, is combined with a DNA fragment contain-
`ing a gene or genes coding for the desired product or
`products.
`The recombinant DNA technique, in its most useful
`form, is based on the following principle:
`DNA can be cut into pieces in a very specific way by
`restriction endonucleases. These pieces can then be
`joined to each other by DNA ligase. DNA cloning
`utilizes a plasmid vector that is a circular DNA mole-
`cule containing only one site for one or several restric-
`tion endonucleases. Treatment of such a vector with a
`restriction enzyme gives one species of a linear mole-
`cule. If this molecule is mixed with a DNA sample that
`is also treated with the same endonuclease, it is possible
`by ligation to obtain molecules composed of the vector
`to which a foreign DNA fragment has been fused.
`These plasmid molecules are called recombinant DNA.
`The vector with the foreign DNA can be transformed
`into a bacterial host cell, which means that it is taken up
`by and replicated in the bacterial host. Since the vector
`is able to replicate, the foreign DNA is also replicated.
`If the foreign DNA is transcribed and translated in
`the bacterial host,
`the gene products of the foreign
`DNA are produced in the bacterial host. This produc-
`tion is in general proportional to the gene concentration
`which, on its side is proportional to the number of cop-
`ies of the recombinant DNA plasmid molecules per cell.
`This means that in order to obtain large quantities of the
`desired gene products of the plasmid, a high number of
`copies of the plasmid per bacterial cell should be aimed
`at. It is known that some cloning vectors inherently
`replicate in a high copy number per bacterial cell, up to
`about 100, However, if the gene product produced by
`the foreign DNA combined with such cloning vector is
`one which is not well tolerated by the bacterial host,
`there may be difficulties in propagating a bacterial clone
`up to the desired production size culture because of
`inhibition exerted by the gene product. On the other
`hand, even a copy number of the order ofabout 20-100
`
`u.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`will not always give rise to satisfactory yield of the
`desired gene product in the production culture. It is
`known that the number of copies of plasmids can be
`further increased by amplifying the inhibition of the
`protein synthesis, for example by addition of chloram-
`phenicol, but as protein synthesis is necessary for pre-
`paring gene products of the cloned DNA, the amplified
`DNA will only be useful for formation of gene products
`thereof if the protein synthesis inhibiting component
`can be removed again, which is not always possible and
`often a complicated procedure.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The present invention provides a process for prepar-
`ing a gene product of a DNA plasmid, in which the
`plasmid is one which allows both effective plasmid
`amplification and obtainment of large quantities of the
`plasmid gene products. The invention utilizes plasmids
`having a temperature-dependent plasmid copy number
`pattern in that the plasmid shows a controlled constant
`plasmid copy number when host bacteria carrying the
`plasmid are cultivated at one temperature, but which
`plasmid shows, when the host bacteria carrying the
`plasmid are grown at a different temperature, an altered
`plasmid copy number pattern allowing a much higher
`or totally uncontrolled copy number. Hence, the num-
`ber of copies of such plasmid is low at one temperature,
`which is an advantage since it decreases the risk that the
`cloned plasmid or its gene products should disturb
`growth of the host bacterium. However, the amount of
`the plasmid can be rapidly increased by a simple tem-
`perature shift, whereby simultaneous formation of the
`cloned plasmid and its gene products is obtained, and
`the production of gene products of the plasmid pro-
`ceeds rapidly.
`the invention provides a
`In accordance with this,
`process for producing a gene product of plasmid DNA,
`said process comprising cultivating bacteria carrying a
`plasmid showing a controlled constant plasmid copy
`number when the host bacteria are cultivated at one
`temperature, and showing, when the host bacteria car-
`rying the plasmid are grown at a different temperature,
`an altered plasmid copy number control allowing a
`much higher or totally uncontrolled copy number;
`under conditions including at least a period of cultiva-
`tion at or approaching a temperature at which the plas-
`mid shows an altered copy number control allowing a
`much higher or totally uncontrolled copy number; and
`harvesting, from the bacterial culture, a gene product of
`the plasmid.
`The crux of the present invention is the utilization of
`the particular type of plasmid having the temperature-
`conditioned plasmid copy number pattern and the rec-
`ognition that this type of plasmid, when copied in a very
`high number at one temperature, also gives rise to pro-
`duction of large amounts of its gene products. The culti-
`vation per se is suitably performed using conventional
`techniques,
`including conventional nutrient media
`which are known to be optimal to the bacterial species
`in question, and also, the harvesting of the gene prod-
`ucts is performed in accordance with well-known meth-
`ods adapted to the identity and properties of the partic-
`ular gene product prepared, the properties of the host
`bacteria, etc. Special and critical to the process of the
`present invention is the temperature regulation involv-
`ing at least a period of cultivation at or approaching a
`temperature at which the plasmid shows an altered
`
`Sanofi/Regeneron Ex. 1033, pg 944
`
`Mylan Ex. 1033, pg 944
`
`

`
`3
`
`4,487,835
`
`4
`dure used to isolate such mutants makes use of the fact
`copy number pattern allowing a much higher or totally
`that the resistance towards ampicillin and chloramphen-
`uncontrolled copy number, in other words, involving a
`icol
`is directly proportional
`to the concentration of
`period of cultivation during which the plasmid is copied
`genes coding for the corresponding resistance enzymes
`in a high number of copies, and, as it has been found,
`(Uhlin et Nordstrom, Plasmid 1, 1977), and of the fact
`during which gene products of the plasmid are formed
`that ampicillin kills only bacteria which are in a phase of
`in correspondingly high amounts.
`growing.
`The plasmid having temperature-dependent plasmid
`It has been found that strains containing plasmid
`copy number pattern may be one prepared by recombi-
`Rldrd-19 (which is described in greater detail below)
`nant DNA technique using a cloning vector showing
`exhibit a single cell resistance on LA plates of about 100
`the temperature-dependent copy number pattern, or the
`pg ampicillin/ml and of about
`100 pg chloram-
`plasmid may be one obtained by mutagenization of an
`phenicol/ml. The resistance in liquid medium towards
`existing plasmid having genes for a desired production.
`these two antibiotics is about the same as on LA plates.
`A detailed description of plasmids showing the
`The resistance towards ampicillin and chloramphenicol
`above-mentioned temperature-dependent plasmid copy
`is directly proportional
`to the gene concentration
`number pattern has not been given previously and ap-
`(Uhlin et Nordstrom, Plasmid 1, 1977). This effect has
`pears from the below examples which describe the
`been used to isolate mutants of plasmid Rldrd-19 with
`preparation of some plasmids of this kind,
`including
`an increased copy number,
`so—called copy mutants.
`plasmids useful as cloning vectors.
`However,
`the combination of a bacteriostatic and a
`The plasmid showing the above-mentioned tempera-
`bacteriocide can also be used to isolate copy mutants
`ture-dependent plasmid copy number pattern with con-
`with different copy numbers at different temperatures,
`trolled constant copy number per cell at one tempera-
`as this selection method allows easy counter-selection
`ture and a much higher or totally uncontrolled copy
`of both parent plasmid and non-conditional copy mu-
`number
`(in the following often termed “runaway-
`tants. The following procedure was found to be a suit-
`replication”) at another temperature may be prepared
`able one: It is assumed that a copy mutant exists with a
`by mutagenization of an existing plasmid which is
`low copy number at a low temperature (30° C.) and a
`known to replicate autonomously in the host bacterium.
`high copy number at a high temperature (40° C.) (the
`in question. In accordance with known principles, the
`procedure is, however, not limited to this type of copy
`mutagenic treatment can be performed in vivo or in
`mutant, but can also be used to isolate other possible
`vitro, and while the mutagenic agents so far used appear
`from the examples, it is assumed that the kind of muta- 30 types of temperature-dependent copy mutants).
`genic treatment
`is not critical. After the mutagenic
`The culture of the plasmid-containing cells is grown
`treatment (and,
`if the mutagenic treatment was per-
`at 30° C., and chloramphenicol is added to a concentra-
`formed in vitro,
`transformation of the mutagenized
`tion (300 ug/ml) which inhibits the growth of cells
`material into the host bacterium), a bacterial clone in
`containing plasmids with normal copy number. The
`which the plasmid copy number is controlled when the 35 cells containing plasmids with a high copy number at
`bacteria are cultivated at one temperature and in which
`30° C. are killed with ampicillin (4000 pg/ml). The
`the plasmid copy number is much higher or totally
`surviving cells are collected and the temperature is
`uncontrolled when the bacteria are grown under differ-
`raised to 40° C. Cells containing plasmids with a high
`ent temperature is then. to be isolated. The isolation
`copy number at 40° C. are selected on ampicillin plates
`procedure may be based upon screening at two temper- 40 (500—2000 pig/ml). Among the cells surviving, some
`should contain temperature-dependent plasmid copy
`atures between which a shift in plasmid number copy
`mutants.
`control pattern is sought for. One indication useful in
`Cells selected in the above manner can then be sub-
`such screening is the fact that on a substrate which is
`jected to further mutagenic treatment, and a suitable
`known to be non-limiting to the host bacterium in ques-
`method for selecting, among the thus mutagenized cells,
`tion, growth inhibition especially at a particular temper-
`a mutant having runaway behaviour at the second tem-
`ature is likely to be due to the production of a large
`perature has been found to comprise an initial selection
`number of copies of plasmid and/or of large amounts of
`for cells which, at the lower temperature, show a con-
`gene products thereof.
`trolled and constant, but increased copy number, indi-
`It has been found that a more suitable way of isolating
`cating that the replication control system has again been
`a plasmid mutant showing runaway-replication pattern
`subject to mutation.
`is often to use a two-stage mutagenic treatment com-
`The particular type of mutation which has occurred
`prising a first stage in which a plasmid-carrying bacte-
`in the specific plasmids illustrated in the examples is not
`rial clone is isolated in which the plasmid shows one
`yet known with certainty, but it
`is believed that an
`plasmid copy number control pattern at one tempera-
`effect of the mutation is that a protein mediated by the
`ture and another plasmid copy number control pattern
`plasmid and involved in the plasmid copy number con-
`permitting a higher copy number at a different tempera-
`trol has, due to the mutation, become modified into a
`ture, and a second stage to obtain, from the mutant
`form which is not stable at the higher temperature.
`obtained from the first stage, a plasmid-carrying bacte-
`While it is evident that the most practical embodiment
`rial clone in which the plasmid shows a much higher or
`of the plasmid of the invention, when it is to be used as
`totally uncontrolled copy number at said second (differ-
`a cloning vector, is one which, like the cloning vectors
`ent) temperature. This procedure allows optimum utili-
`described in the examples, in itself contains all elements
`,zation of suitable selection techniques, for example dou-
`ble antibiotic selection:
`necessary for the temperature-dependent copy number
`behaviour. it is also obvious that the principles of the
`One type of plasmid copy mutants has the following
`present
`invention and advantages conferred thereby
`characteristics: At low temperature (e.g. 30° C.),
`the
`may also manifest themselves when the mutation is of
`copy number is close to that of the parent plasmid,
`the nonsense type combined with corresponding tem-
`while at higher temperature (e.g. 40° C.), the copy num-
`perature-dependent nonsense suppressor in the host
`ber is about 4-fold higher. A suitable isolation proce-
`
`10
`
`15
`
`20
`
`45
`
`50
`
`55
`
`60
`
`Sanofi/Regeneron Ex. 1033, pg 945
`
`Mylan Ex. 1033, pg 945
`
`

`
`4,487,835
`
`5
`bacterium, for example when the plasmid is an amber
`mutant and the host bacterium is one which shows
`temperature-dependent amber suppressor effect.
`The temperature at which the plasmid shows run-
`away behaviour will not necessarily be one which is
`higher than the temperature at which the plasmid shows
`a controlled constant copy number, also the reverse
`situation is possible. If it is desired to prepare a mutant
`showing runaway replication at alower temperature
`than the temperature giving controlled constant copy
`number, the selection or screening criteria are adapted
`correspondingly, However, when the gene product to
`be prepared by cultivating the plasmid-containing bac-
`teria is one which is not deteriorated at the higher tem-
`perature, it is preferred that the temperature at which
`the plasmid shows runaway replication is a higher tem-
`perature than the one giving a controlled constant plas-
`mid copy number. When this applies, the amplification
`of the plasmid takes place under the same conditions
`allowing the relatively highest cell growth rate. The
`plasmids illustrated in the working examples were de-
`signed to show, in the mesophilic bacteria exemplified
`by Escherichia coli, a constant controlled plasmid copy
`number at 30° C., and runaway behaviour at 40° C. As
`will appear from the data on these plasmids, the con-
`stant controlled plasmid copy number is retained up to
`about 32° C. for these plasmids and the temperature at
`which the plasmid shows runaway behaviour is above
`about 36° C.
`The most interesting plasmids of the present inven-
`tion are plasmids which show a reasonably large, but
`constant copy number at one temperature, and a copy
`number which is at least 20 times higher at the different
`temperature. Whether or not total runaway behaviour
`with resulting death of the cell is obtained will to some
`extent depend upon the bacterial host and its ability to
`tolerate the plasmid and its gene products in high con-
`centration.
`the present invention permits, on the one
`Hence,
`hand, obtainment of plasmid copy numbers which were
`hitherto unobtainable, up to the order of several thou-
`sand per cell, and, as it has been found, ‘concomitant
`high production of gene products of the plasmid during
`a sufficient number of generations (usually 4-6) of
`growth at the high copy number to secure a consider-
`able production of gene products. On the other hand,
`the invention provides a most simple control of the
`copy number, which may be utilized in various man-
`ners, depending upon the individual conditions with
`respect to bacterial host, desired gene product, etc.: In
`most cases, it will be preferred that the propagation of
`the bacteria from an individuum or a clone up to a
`production size culture is performed at or nearthe tem-
`perature at which the plasmid shows a controlled con-
`stant copy number, in order to avoid any inhibition of
`the bacterial growth by an increasing plasmid and gene
`product concentration. Thereafter,
`the temperature
`may be shifted to a temperature at which the plasmid
`shows an altered copy number control allowing a much
`higher or totally uncontrolled copy number, and after a
`suitable production period, often until the growth of the
`bacteria is inhibited by the production of the plasmid
`and/or gene products thereof,
`the harvesting of the
`gene product is performed. Depending on the particular
`conditions, it may be desired to perform the production
`cultivation at a temperature which is only approaching
`the temperature at which the plasmid shows the altered
`copy number control, so as to obtain a steady-state
`
`l0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6
`culture giving a high yield of gene product (which may
`then be continuously or intermittently harvested from
`the culture in a manner known per se), but at which
`temperature the host cells survive and are still capable
`of propagation.
`One example of the unique control possibilities ob-
`tained through the present invention is preparation of a
`temperature-sensitive protein as the desired gene prod-
`uct. In such case, the bacterium may be propagated up
`to production size culture at the temperature at which
`the plasmid copy number is controlled and constant,
`and thereafter, by a temperature shift to the runaway
`temperature, the plasmids may be amplified. Subsequent
`to amplification of the plasmids and while the cells are
`still viable and capable of propagation, the temperature
`is again shifted to the lower temperature giving con-
`stant copy number, and at this lower temperature, the
`resulting bacteria carrying the much higher number of
`plasmids per cell are used for the production of the
`temperature-sensitive protein in question. It is known
`that there are many organisms which are not viable at
`temperatures above about 30° C., and the proteins of
`which, therefore, may be denatured at higher tempera-
`tures. Therefore, this embodiment of the temperature
`regulation may prove especially important when gene
`products inherent to such organisms are to be prepared
`by recombinant DNA/cloning technique.
`As indicated above, the plasmid showing the charac-
`teristic temperature-dependent copy number behaviour
`involving runaway replication may be one derived by
`mutagenic treatment, from a parent plasmid possessing
`genes for the desired production, but without the char-
`acteristic runaway behaviour. However, for many prac-
`tical purposes the plasmid used in the production culti-
`vation will be one prepared by recombinant DNA tech-
`nique, using as a cloning vector a suitable plasmid show-
`ing the characteristic temperature-dependent runaway
`replication. This combines the advantages of the con-
`ventional recombinant DNA technique with the advan-
`tages of the temperature control and the very high
`obtainable number of plasmid copies per cell, with the
`consequent amplified production of polypeptide or pro-
`tein mediated by the foreign DNA fragment. It is con-
`templated that the very high number of plasmid copies
`obtainable by using a cloning vector of the present
`invention will permit production of large or at any rate
`reasonable amounts of proteins which, due to their
`being genetically relatively remote from the host bacte-
`rium in question, could hitherto not be produced at all,
`or at any rate not be produced in a satisfactory amount,
`by cloning techniques. Hence, the cloning vector aspect
`of the present invention is a very important aspect.
`In order to be useful as a cloning vector, the plasmid
`should show, for at least one restriction endonuclease,
`one and only one site susceptible to the endonuclease,
`and the said site should be one which after insertion of
`a fragment of foreign DNA at this site, permits the
`resulting recombinant DNA to replicate autonomously
`and, to obtain the advantages of the present invention,
`with retention of the capability of showing the tempera-
`ture-dependent copy number pattern.
`The most suitable restriction endonucleases for use in
`recombinant DNA technique are those giving the so-
`called “cohesive ends" on both the cloning vector and
`the DNA fragment,
`in other words, single stranded
`regions at the ends of the molecules with complemen-
`tary base sequence allowing base pairing to identical
`sequences.
`
`Sanofi/Regeneron Ex. 1033, pg 946
`
`Mylan Ex. 1033, pg 946
`
`

`
`4,487,835
`
`8
`7
`techniques used were standard
`The experimental
`As appears from the examples, cloning vectors hav-
`techniques used in microbial genetics (J. Miller, Meth-
`ing one site for restriction endonuclease have been pre-
`ods in Molecular Biology, Cold Spring Harbor Labora-
`pared, and also vectors having one site for one restric-
`tion endonuclease, and another site for another restric-
`tory) and in genetic engineering (T. Tanaka and B.
`5
`Weissblum, J. Bacteriol. 212 (1975) 354-362).
`tion endonuclease, has been prepared. An advantageous
`TABLE 1
`Esc/zerichin colt‘ K-l2 strains
` Strain Genotype Reference or Source
`
`
`Ecloos
`thi, met, nal, relA
`Grindsted et al, J. Bact. Ill) (1972) 529.
`Dll
`thi, his, pro, trp, strA
`Boman et al. Genet. Res. 12 (1968) 169.
`C600
`thi. lac. leu. thr
`Appleyard, R. K., Genetics 39 (1954) 440.
`UB173l
`thi, met, pro, amp (Tn802), rial
`Bennett, P. M. and M. H. Richmond, J. Bact. 126
`(1975) 1.
`
`H00
`
`thi, endol
`
`TABLE 2
`Plasmids used
`Source "T
`‘
`MCVW“ D3113. Nature 214 (1967) 335.
`5° 9‘ 31» 3‘ sen Genet. 142 (1975) 239.
`
`Plasmid
`
`R'd"“9
`PSF2124
`
`way of constructing cloning vectors fulfilling the above
`conditions is often to prepare, from a larger plasrnid
`showing the temperature—dependent runaway replica-
`tion, a “miniplasmid" of sufficiently small size to show 20
`only one site susceptible to a useful restriction endonu-
`clease but with sufficient size to still contain the genes
`indispensable to the special temperature-dependent rep-
`lication behaviour. Such miniplasmids may be prepared
`S
`P
`Y
`S
`Plasmid R1 is a transferable plasmid that mediates
`from a lar er
`lasmid b isolatin bacterial clones car- 25
`rying either spontaneously occurring or in vitro-pre-
`resistance to the following antibiotics: ampicillin, chlo-
`ramphenicol, kanamycin, streptomycin, and sulphona-
`pared (and thereafter transformed) miniplasmid deriva-
`tives of the parent plasmid. The isolation of the desired
`mides. The plasmid has a molecular weight of 65X 105
`daltons. Normally,
`in Escherichia coli,
`the plasmid is
`miniplasmids (or bacteria containing them) is performed
`by suitable and well-known screening and/or selection 30 present in about one copy per chromosome equivalent.
`methods.
`It is possible by mutation in this plasmid to increase the
`In principle, an ideal cloning vector is one which
`copy number several fold above this level, so-called
`contains as few genes as possible coding for the produc-
`copy mutants (K. Nordstrom et al., J. Bacteriol. 110,
`tion of non-desired proteins. However, for many pur-
`562-569 (1972)).
`poses,
`it
`is desired that
`the cloning vector contains 35
`In the examples, the following stages are described:
`genes mediating a so-called marker useful for identifica-
`Example l: Isolation of a plasmid mutant (pKN30l)
`tion and/or selection of cells carrying the plasmid. The
`which has a temperature—dependent
`replication
`most useful marker is antibiotic resistance, for example
`control. The copy number of this plasmid increases
`ampicillin resistance, as this permits, after a treatment
`about 4 fold at higher temperature.
`for transforming a recombinant DNA into a bacterial 40
`Example 2: The isolation from plasmid pKN30l of a
`host, an easy counter-selection of bacteria which have
`mutant (pKN400) that lacks replication control at
`not received the recombinant plasmid. However, also
`higher temperature (a runaway mutant).
`cloning vectors without a marker may be useful cloning
`Example 3: The isolation from plasmid pKN4OO of a
`vectors, for example when the genes to be inserted by
`miniplasmid (pKN402) that retains the runaway
`the recombination in themselves carry a marker.
`behaviour but carries only one site for the restric-
`When it is desired to introduce a marker, for example
`tion endonuclease EcoRl.
`antibiotic resistance, in a plasmid of the present inven-
`Example 4: The isolation from a derivative of plasmid
`tion to be used as a cloning vector. this may be done by
`pKN4OO of another miniplasmid that retains the
`transposition of a DNA fragment in a