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`SANOFI V. GENENTEC l
`SANOFI v. GENENTECH
`IPR2015-01624
`31.__ IPR2015—0162
`EXHIBIT 2035
`EXHIBIT 203 ‘
`,..
`_
`3.2’.
`
`1
`if US. GOVERNMENT PFIINWNG OFFICE: 1930322322
`
`»._\.
`1;‘.
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`
`CONTENTS
`
`Xi'1.Apph'
`
`'ons .-:__pa_rpers.
`
`1,’,
`
`¥_____
`
`
`
`1
`
`
`
`United States Patent
`
`[19]
`
`[:1] Patent Number:
`
`4,495,280
`
`Bujard et al.
`
`[-15} Date of Patent:
`
`Jan. 22, 1985
`
`[75]
`
`[54] CLONED HIGH SIGNAL STRENGTH
`PROMOTERS
`Inventors: Hermann G. Bnjard, Heidelberg.
`Fed_ Rep of Germany; Annie C_ Y_
`Chang’ P310 Alto: Stanley N_ Cohen,
`P011013 V3115?’ both of Calif‘
`[73] Assignee: The Board of Trustees of the Leland
`Stanford Jr. University, Stanford,
`Calif.
`
`_
`[2]] App!" No" 265316
`[22] Filed:
`May 20. 1981
`[51]
`Int CL; _______________________H CIZQ 1/63;C12Q 1/02;
`
`'
`
`_______________ _l 435/5; 435/29:
`[52] U‘s_ CL _____________________
`435,33; 435/}‘_!2_3: 435/91; 535/37; :;-35;(,;
`935/35; 935/37: 935/41; 935,115
`Fiend gf srch ,,,,,,,,,,,,,,,,,,,,, __ 435/29, (,3, 5, 172,
`435/240. 253. 254. 257. 253. 317, 91; 536/27,
`23. 29
`
`[sa]
`
`[56]
`
`References Cited
`PUBLICAIIONS
`Stuber et al., PNAS, 73(1).
`l6'r'—l71. (Jan. 1981).
`
`Casadaban et al., J. Mol. 3501., 133. 179-207, (1930).
`West at 31.. Gene. 9. 175-193. (1980).
`Gfibain ‘it 3'3" PNA5- mm‘ 139493 U979)‘
`Stuber et al., Moles. Gen. C'renet.. 166, 14l—l4-9. (1978).
`Stiiher et al., PNAS USA. 73(1), 167-171, (1931).
`Primary Exami'ner—Esther M. Kepplinger
`Attorney. Agent. or Ft‘rm—Berlram 1. Rowland
`
`ABSTRACT
`[57]
`Method for preparing high signal strength promoters
`and terminators and DNA compositions employing
`such_prornoters and terminaiors. 5 phage is cleaved to
`provide for DNA sequences having ll'lIi1CI promoters.
`T115593 pl'OI'I10[E'l'S 3T|.’:'
`iflfifirlfid ill“) \I"CI?[0l'.‘i Sflpflffltfid
`from a balanced terminator by 2 gene ofmteresl and the
`terminator is desirably followed by a mzirker allowing
`for selection of Iransformants. High efficiencies In tran-
`Scripiion of DNA can be achieved with the highly ac-
`live TS promoters. The promoters and terminalors are
`used in hybrid DNA for efficient expression of struc-
`tural genes and transcription to provide RNA sequen-
`Gas
`
`'
`
`15 Claims, 1 Drawing Figure
`
`2
`
`
`
`
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`fen ffd}
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`cm‘
`
`Hpaflf DNA Pol.
`{vividIIIEcofll
`Dj—.
`
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`pBR322
`IECDRIIDNA Pol. /I-khd III}
`
`Hr}.-CI!
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`
`——u:——-——-
`i
`P/0 ‘.5 -gal
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`DNA Pol.
`Liguse
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`{fifrrd III}
`
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`
`
`
`CLONED HIGH SIGNAL STRENGTH
`PROMOTERS
`
`The government has rights in the invention pursuant 5
`to Grant Nos. AI 08619 and GM 27241 awarded by the
`National Institute of Health.
`
`The research was supported in part by a grant from
`the Deutsche Forschungsgemeinschaft.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`20
`
`25
`
`JD
`
`1. Field of the Invention
`Having established the feasibility of producing a wide
`variety of naturally occurring and synthetic polypep- .
`l5
`tides by means of hybrid DNA technology. there are
`continuing and extensive efforts to provide for more
`efficient and economic methods for producing the poly-
`peptides. In developing a process for the commercial
`production of polypeptides. many factors will be in-
`volved in optimizing the economic and efficient pro-
`duction of the polypeptides. Included among these fac-
`tors are regulatory signals. which are DNA sequences
`involved with the regulation of replicaton. transcription
`and translation.
`One area of interest is at the level of transcription.
`Transcription involves the enzyme RNA polymerase.
`The RNA polymerase binds to a site qlled a promoter.
`It has been observed that promoters vary in their activ-
`ity. as evidenced by the number of initiations of RNA
`per unit time or the strength of binding of the enzyme to
`the promoter site. The promoter may have one or more
`sequences that bind. which may or may not be contigu-
`ous. The more active promoters are referred to as
`strong promoters-
`It was Found that when introducing a strong pro-
`moter into a vector and employing the resulting plasmid
`for transformation. one could not select transformants
`based on expression of markers which allowed for selec-
`tion. Therefore, cloning of the strong promoters was
`not feasible. It is therefore desirable that methods be
`provided which would allow for The screening of strong
`promoters and terminators and their subsequent cloning
`to be used in conjunction with the replication, transcrip-
`tion and translation of the genes for production of 45
`DNA. RNA, and polypeptides.
`2. Description of the Prior Art
`Promoters from bacterial and viral sources have been
`cloned in E. calf. and their signal strength in vitro has
`been studied using expression from distal promoterless
`sequences encoding .3-galactosldase or other proteins
`(Casadaban and Cohen (1980) J. Mol. Biol 138, 179-207;
`West and Rodriguez (1930) Gene 9, l'l5—l93). Attempts
`to clone small DNA fragments carrying the strong
`promoters of bacteriophage T5 have been unsuccessful
`(v. Gabain and Bujartl ([9791 PNAS USA 76. 189. 193).
`Fragments of T5 DNA having both a strong promoter
`and a strong termination signal have been cloned.
`(Brcunig (1979) Dissertation (Universitat Heidelberg.
`Heidelberg, Germany)) Analysis has shown that tran- no
`scriptional regions of several E. roll" plasmids are orga-
`nized in units where initiation and termination signals.
`are balanced. (Stuber and Bujard (I98 1] PNAS USA ‘.-'3:
`157-171) P15 and P25. promoters of the T5 bacteriophage
`are reported as among the most efficient RNA polymer-
`ase binding sequences. (Stuber et al ([973) Moi. gen.
`Genet.
`166 141-149; Niemann (1981) Diplomarbeit
`(Universitat Heidelberg. Heidelberg. Germany)}.
`
`35
`
`50
`
`55
`
`1
`
`4,49 5,2 80
`
`2
`SUMMARY OF THIE. INVENTION
`
`Methods for cloning. sequencing and using strong
`promoters and terminatots are provided. as well as
`corupositions resulting from the methods. By cleaving
`T5 phage and selecting fragments specifically binding
`to RNA polymerase, fragments containing promoters
`are isolated. A vector is constructed having a strong
`promoter. followed by a DNA sequence of interest,
`optionally followed by one or more translational stop
`codons in one or more reading frames, followed by a
`balanced terminator, followed by a marker allowing for
`selection of transformants. Upon introducing the result-
`ing plasmid into a microorganism host, efficient tran-
`scription of the gene is obtained with substantially lesser
`expression of the market as compared to the gene. The
`level of expression of the marker permits selection of
`transformants having the above described construct.
`The construct or regulatory portions thereof are used
`for efiicient transcription of RNA or gene expression.
`BRIEF DESCRIPTION OF THE DRAWING
`
`FIG. 1 is a How chart of the preparation of a plasmid
`for cloning strong promoters and terminators.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`Methods and compositions are provided for prepar-
`ing and cloning strong promoter and terminator regula-
`tory signals and utilization of the strong regulatory
`sequences in the transcription and expression of genes of
`interest.
`Particularly, T5 phage promoters are isolated, cloned
`in conjunction with a strong terminator, and appropri-
`ate vectors developed for insertion of DNA sequences
`of interest, usually structual genes. to provide for high
`and efficient
`transcription and/or expression of the
`sequence.
`The compositions of this invention are characterized
`as having in the downstream direction of transcription
`the following units: a strong T5 phage promoter; op-
`tionally a structural gene which may be a marker; a
`balancing terminator; and optionally a marker allowing
`for selection of transfonnants containing the construct.
`which marker has a relatively low level of expression in
`comparison with the amount of RNA polymerase initia-
`tion at the promoter. In the absence of a promoter in the
`construct. the construct can be used for the cloning and
`characterization of promoters of different strengths.
`In referring to strong promoters, it is intended that
`the binding affinity of RNA polymerase is stronger than
`the commonly employed promoters such as lac and trp
`and at least comparable to and normally greater than
`the combination of lac and trp promoters. For the most
`part. the strongest promoters among prokaryotes are
`the T5 phage promoters and these will be employed as
`exemplary of naturally occurring or synthetic strong
`promoters. It is to be understood. that other prokaryotic
`and euknryotic promoters. either naturally occurring or
`synthetic. could find application in the subject inven-
`Lion.
`
`The compositions of this invention will include linear
`segments for insertion of DNA having the strong regu-
`latory signal sequences (i.e.. the promoter and termina-
`tor) adjacent opposite ends of the linear segment and
`plasmids formed by introducing a DNA sequence from
`a source other than the source of the promoter as a
`bridge between the strong regulatory signal sequences.
`
`4
`
`
`
`3
`The termini may be blunt or staggered ended, having
`the same or different termini to allow for directed posi-
`tioning of inserted sequences.
`The compositions which are employed as already
`indicated have a promoter, a balanced terminator, and
`desirably a marker which are in the direction of tran-
`scription when the signal sequences are joined. The
`terminator is balanced, so that a small but useful amount
`of expression of the marker can occur. Normally. in
`most situations, a gene will bridge the promoter and
`terminator to provide circular DNA. The gene may he
`a non—st.ructu1'al gene or a structural gene providing
`RNA e.g.
`ribosomal or messenger. or providing a
`poIy(an:u’no acid).
`.
`Desirably, the gene is followed by one or a plurality
`of translational stop codons e.g. oop or nonsense co-
`dons. or preferably a plurality. usually up to about six.
`more usually from about two to five, where there is at
`least one stop codon in each reading frame. The stop
`codons aid in the efficiency of termination, both at the
`level of transcription and expression.
`Next in the direction of transcription is the terminator
`sequence. The terminator sequence is balanced with the
`promoter in the sense that the marker is transcribed not
`more than about one-fourth of the times that a gene
`upstream from the terminator is transcribed. where both
`genes are under the control of the same promoter._ Usu-
`ally. it will be desirable that a sufficient amount of the
`marker is transcribed to allow for selection of transfor-
`mants. In view of the fact that the marker is transcribed
`from a strong promoter, it will usually be sufficient that
`the marker is transcribed in relation to the transcription
`of the gene intermediate the promoter and terminator in
`only about 1 to 20, usually only 5 to 15 number % ofthe
`gene. The strength of the promoter is reflected in the
`level of expression of the marker which is transcribed
`from the same promoter as the gene whose expression is
`desired.
`In addition to the DNA sequences indicated above.
`there will normally be other regulatory signals neces-
`sary for expression involved with the DNA sequence.
`such as translational start and stop sites. In addition to
`the foregoing regulatory signals, other regulatory sig-
`nals may be included. such as additional promoters.
`operators. initiators, catabolite activator protein binding
`sites, etc. Furthermore, the promoter and terminator
`may be separated by more than one gene.
`that is, a
`plurality of genes, including multimers and operons.
`The above DNA sequence construct will have a
`replicating system or be cleaved and be inserted into a
`vector to provide a plasmid. The vector is distinguished
`by having one or more DNA sequences which serve to
`insure stable replication of the plasmid and may also
`provide opportunities for high copy numbers of the
`plasmid in the microorganism host. The vectors may be
`derived from chromosomal or extrachromosomal
`sources. The sources include plasmids, viruses (phage).
`chromosomes, or the like. In addition. the vector or the
`essential portions thereof may be prepared syntheti-
`cally.
`The plasmids may then be used for transformation of
`an appropriate microorganism host. Methods of intro-
`ducing DNA into an appropriate host are well known.
`Illustrative of such methods. but not exhaustive of such
`methods, are transformation e.g. calcium shock. trans-
`fection, and conjugation. Descriptions of these methods
`may be found in Genetic Engineering, ed. Setlaw and
`Hollaender, Vol. 1. Plenum Press, New York and Lon-
`
`IO
`
`l5
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`SS
`
`60
`
`65
`
`4,495,230
`
`4
`don_. 1979; Molecular Cloning of Recombinant DNA,
`ed. Scott and Werner, Vol. 13, Academic Press, Inc.
`New York. I973, and references cited therein.
`In order to allow for flexibility in preparing the con-
`struct and self-replicating sequence or plasmid contain-
`ing the construct restriction sites should be present to
`allow for unique insertions and isolation of‘ the various
`elements. The restriction sites may be naturally present.
`introduced by linkers. result by partial sequential nucle-
`otide removal from a chain using an exonuclease, or the
`like. Desirably,
`the restriction sites will provide for
`different ends to permit only the proper orientation of
`the inserted fragment.
`A wide variety of structural genes are of interest for
`production of proteins,
`including but not
`limited to
`proteins of physiological interest. proteins as chemicals,
`and enzymes which may be of direct interest or of inter-
`est
`in transforming another product, which may be
`proteinaceous or non-proteinaceous. The proteins may
`be prepared as a single unit or as individual subunits and
`then joined together in appropriate ways. Furthermore,
`as appropriate, the protein products may be modified by
`giycosylation. acylation with aliphatic acids. e.g. lipid
`acids. phosphorolation. sulfonation or
`the like. The
`different classes of proteins which may be prepared
`include protamines. histones. alburnins globulins. sclero-
`proteins. phosphoproteins, rnucoprotelns. chromopro-
`teins, lipoproteins, nucleoproteins. and the remaining
`proteins which are unclassified.
`The following is a representative list of proteins of
`interest.
`_
`leukocyte.
`interferon e.g.
`Insulin; growth hormone;
`lumphoblastoid, T-immune and fibroblast; ,3-lipotro~
`pin; £3-endorphin; dynorphin; histocompatability pro-
`teins; immunoglobulins e.g. lgA. lgD. IgE. IgG and
`IgM and fragments thereof; hemoglobin. somatome-
`dins;
`lympholtines; growth factors e.g. epidermal.
`fibroblast. platelet-derived. multiplication stimulating
`and nerve; hematoporetic-stimulating factors e.g.
`erythropoietin, colony-stimulating. erythroid poten-
`tating activity or burst-promoting activity and lym-
`phopoietins; albumin and prealbumin;
`Prcalburnin
`Albumin
`at-Lipoprotein
`a|—Acid glycoprotein
`at-Antitrypsin
`or-Glycoprotein
`Transeonin
`' 4.68-Postalbumin
`Tryptophan-poor
`rt;-glycoprotein
`rt;-X-Glycoprotein
`Thyroxin-binding globulin
`Inter-ct-trypsin-inhibitor
`Gc-globulin:
`(Gc I-1).
`(Gr: 2-1).
`(Gc 2-2).
`I-iaptoglobin:
`1-I)!
`(Hp 2-1).
`2:2):
`Ccruloplasrnin
`Cholinesterase
`oz-Lipoprotein(s)
`ctz-Macroglobulin
`otz-HS-Glycoprotein
`
`5
`
`
`
`5
`
`4,495,280
`
`6
`
`Zn-ag-glycoprotein
`cg-Neuramino-glycoprotein
`Erythropoietin
`B-lipoprotein
`Transferrin
`Hernopexin
`Fibrinogen
`Plasruinogen
`ct;-glycoprotein I
`dz-glycoprotein II
`Immunoglobulin G
`636) or ')-‘G-globulin
`Mol. formula:
`‘?2K2 0!‘ 7212
`Immunoglobulin A (IgA) or 'yA-globulin
`Mo]. formula:
`
`(a2~:zl" 0r (u2?t2)"
`Irnniunoglobuljn M (IgM) or 7M-globulin
`Mo]. formula:
`
`0L2I¢2)5 OI‘ (ll-23t2)5
`Irnmunoglobulin D (IgD) or 'yD-Globulin ('yD)
`Mot. formula:
`(-52x2) or (5211)
`Immunoglobulin E (IgE) or 'yE-Cilohttlin (713)
`M01. formula:
`(GM) of (£212)
`Free light chains
`Complement factors:
`C’1
`C'lq
`C'1r
`C‘ls
`C2
`03
`31A
`
`cr.1D
`C4
`C5
`C6
`C".-'
`C'3
`C'9.
`
`Important protein hormones include:
`
`Peptide and Protein Hormones
`
`Parathyroid hormone: (parathormone)
`Thyrocalcitonin
`Insulin
`
`Glucagon
`Relaxin
`Erythropoietin
`Melanotropin: [Mehinocyte-stimulating honnone; inter-
`medin)
`Somatotropin: (growth hormone)
`Corticotropin: (adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Leuteinizing hormone: (interstitial cell-simulating hor-
`mone)
`Lutcomammotropic hormone: (Luteotropin. prolactin)
`Gonadotropin: (chorionic gonadotropin).
`Tissue Hormones
`
`Secretin
`Ciastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactogen
`
`Peptide Hormones from the Neurohypophysis
`
`Oxytocin
`Vasopressin
`Releasing factors (RF): CRF, LRF, TRF, Somatotro—
`pin-RF, GRF, FSHRF, PIF, MIF.
`
`In addition to various non-enzymatic proteins of
`physiological interest. enzymes can also be produced as
`an end product or for intracellular transformation of a
`substrate present in the host or substrate introduced
`eittracellularly, or for enzymatic transformation in vi-
`tro.
`In accordance with the I.U.B. classification, the en-
`zymes fall into varying categories such as 1. oitidore-
`ductases; 2. transferases; 3. hydrolaaes; 4. lyases; 5. isom-
`erases; 6. ligases. Enzymes of particular interest will be
`hydrolases and oxidoreductascs for use in commercial
`processing. for example. hydrolases for hydrolysing
`polysaccharides, lipids and polypeptides; oxidoreduc—
`tases for oxidation of alcohols and aldehydes. epoxida-
`tion. and the like.
`The microorganism host may be bacteria, such as
`Escherichia. Bacillus, Aerobacter. Klebsiella, Proteus.
`Pseudomonas, Streptococcus, Staphylococcus. Clo-
`stridiurn, Mycobacteriurn, Streptornyces and Amino-
`rnyces; Fungi e.g. Gymnomycota, Dimastygomycota,
`Eumycota. Zygomycetes. Ascomycetes and Ba5idorny-
`cetes, such as. Candida, Aspergillus, Rhizobus, Micro-
`sporum. and Fonsecaea; Protozoa e.g. Mastigophora.
`Sarcodina. Sporozoa and Celiophora. such as, Trypano-
`soma, Codosiga, Protospongra and Entarneba, and Alga
`e.g. Dinoflagellates. Euglenoids, and Diatorns.
`Higher cells. e. g.. mammalian, may also be employed
`as hosts, where viral. e.g.. bovine papilloma virus or
`other DNA sequence is available which has plasmid-
`like activity-
`Depending upon the nature of the host. a wide vari-
`ety of vectors may be employed. The vector will have
`an intact replicon and be capable of replication in the
`host. In addition, replicons can be developed which
`may have one or more other regulatory signals. Regula-
`tory signals can include temperature sensitive replicons,
`runaway-replication sequences.
`temperature sensitive
`operators, and the like. Various additional DNA se-
`quences may be present providing for restriction sites.
`markers, termination sequences. or the like. Desirably.
`the vector should be of a substantially difierent size
`from the construct to allow for excision of the construct
`and ease of separation of the construct from the vector
`by molecular weight separation techniques e.g. electro-
`phoresis and density gradient centrifugation.
`The subject invention also provides for a technique
`for screening the strength of promoters and ten-ninators.
`thus allowing for the determination of the use of a pro-
`moter or terminator in a particular application, where it
`may be desirable to have promoters or terrninators or
`combinations thereof‘ of varying strength. In screening
`promoters and/or terminators one empioys a promoter
`or terrninator of known activity. A DNA construct is
`made having the sequence described previously. where
`appropriate restrictionsites are provided for introduc-
`ing the various elements. The construct provides in the
`direction of transcription the promoter. a first gene
`marker. optionally termination codons. such as non-
`sense codons and oop terminator, the terminator, and a
`second gene marker. This DNA construct is inserted
`into an appropriate vector. Where the promoter and
`
`10
`
`15
`
`I5
`
`30
`
`35
`
`45
`
`SD
`
`55
`
`65
`
`6
`
`
`
`4,495,280
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`S5
`
`7
`terminator are properly balanced, there will be a sub-
`stantial differentiation between expression of the first
`gene marker and expression of the second gene marker.
`The ratio of expression between the first and second
`marker will provide for a comparative evaluation of the 5
`activity of the promoter or terminator, depending upon
`which is of known value. Thus, one can degrade a DNA
`sequence such as a chromosome. or an extrachromo-
`sorual element, such. as a-plasmid or double minute,
`isolate the promoters by selective binding with RNA
`polymerase and insert the DNA fragments which bind
`to the RNA polymerase into the previously described
`construction. By detennining the relative proportion of
`expression of the first and second gene markers. one can
`determine the strength of promoters in relation to a
`fixed terminator. Similarly, one can isolate DNA se-
`quences having terminator sequences, insert the sequen-
`ces into the above described construct at the appropri-
`ate site and then measure the relative expression of the
`two gene markers.
`Various markers can be chosen for evaluating the
`relative activities of promoters and terrninators. Conve-
`niently. markers which allow for selection such as resis-
`tance to antibiotics. toxins or heavy metals can be used.
`By varying the concentration of the selective agents in
`the nutrient medium. one. can determine the relative
`proportions of the enzyme expressed by the genes in
`relation to the growth of the host. Alternatively, one
`can use growth factors e. g. having a gene which com-
`plements a mutant gene in an auxotrophic host. where
`the gene expresses a product necessary for a blown-
`thetic pathway. A third marker provides virus incom-
`patibility, preventing plaque formation. Other markers
`which allow for comparison will come readily to mind.
`The terminators may be evaluated in the presence
`and absence of rho. so that one can determine the de-
`pendency of the terminator on tho, as well as the effec-
`tiveness of the terminator in relation to the concentra-
`tion of rho.
`The methods for preparing the subject compositions
`will be conventionaL The various DNA fragments and
`sequences can be obtained from a variety of sources by
`restriction mapping and endonuclcase cleavage to pro-
`vide fragments having the desired intact sequence or
`gene. The fragments can be further processed employ-
`ing endo- or exonucleases to remove nucleotides unre-
`lated to desired regulatory sequences or structural
`genes. By appropriate choice of restriction enzymes.
`cohesive or blunt ended fragments can be generated.
`Furthermore. chains can be extended with single nucle-
`otides or oligonucleotides.
`linkers can be added, or
`otherwise processing to provide for termini having
`desired properties.
`Desirably. a vector is employed having appropriate
`restriction sites, a competent replication system for the
`intended host. and optionally one or more markers
`which allow for selection. For hybrid DNA technology
`it would be useful to have a plasmid having a unique
`restriction site between a T5 promoter and a terminator.
`desirably having at
`least one stop codon on the up-
`stream side of the terminator. In this manner. one or
`more structural genes may be introduced between the
`promoter and terminator.
`A5 appropriate, downstream from the promoter. but
`remaining proximal to the promoter, may be an opera-
`tor. activator. ribosornal start signal sequence. or the
`like. to allow for controlled expression of the inserted
`gene(s).
`
`65
`
`8
`The strategy described above provides a vehicle
`which can be used with one or more hosts for gene
`expression, where the gene after processing in a prede-
`termined way can be directly inserted into the vehicle
`to provide a competent plasmid for expression of the
`desired gene(s).
`Alternatively. the gene(s) of interest may be ligated
`to the appropriate regulatory signal sequences before
`insertion into the vehicle. In this instance, only the
`promoter and terminator regulatory signals need be
`present.
`To provide for enhanced flexibility, the region be-
`tween the promoter and terminator may be designed so
`as to provide for a plurality of restriction cleavage sites,
`allowing for the introduction and removal of DNA
`fragments without interruption of the remainder of the
`vehicle. Thus, by having a plurality of unique restric-
`tion sites or restriction sites limited to the region be-
`tween the promoter and terminator in the downstream
`direction of transcription, regulatory signals and genes
`may be readily inserted and removed.
`Another strategy is to prepare a construct having all
`of the desired DNA sequences for transcription and
`expression in appropriate sequence. with the construct
`having predetermined termini and inserting the con-
`struct into an appropriate vector which has been linear-
`ized to provide complementary termini.
`In developing the construct, a vector will normally
`be used in order to clone the various sequences. The
`construct will allow for the insertion of the different
`sequences in the correct direction and desirably only in
`the proper orientation. Therefore,
`it will usually be
`desirable to have the sequence and insertion site be
`asymmetric in having different termini with the termini
`of the sequence and insertion site being complementary.
`The particular restriction enzymes will vary widely
`with the various sequences. there being a large number
`of restriction enzymes of known base or sequence
`specificities commercially available.
`The following examples are offered by way of illus-
`tration and not by way of limitation.
`EXPERIMENTAL
`
`Materials and Methods
`I-Ilncll. Sall,
`Restriction endonucleases Hindlll,
`BamHI and T4 Ligase were from New England Biolabs
`(Beverly. Mass, USA). Sau3A was from BRL (Neu-
`Isenburg. Germany) and EcoRI and HpaII from (Bo-
`ehringer Mannheim, Federal Republic of Germany).
`DNA polymerase I. phage fd DNA (replicative form}.
`plasmid AD16/30 containing a 28 bp Hindlll/Batnlll
`adapter sequence and. lac represser were supplied by
`private sources. The isolation of bacteriophage T5
`DNA, plasmid DNA E. coli RNA polymerase and E.
`colt‘ termination factor rho have been described previ-
`ously, {v. Ciabian and Bujard. Moi. gen. Genet. (1977)
`[57, 301-311; Clewel and Helinski (1969) PNAS USA
`62, 1157-1166; and Knopf and Bujard (1975) Eur. J.
`Biochem. 53, 371-385).
`A 'l3Ubp DNA fragment carrying the E. coil’ lac regu-
`latory region (promoter/operator: P/O), an N-terminal
`portion of the .Cl-galactosidase structural gene sufficient
`for intracistronic complernentation of the M15 deletion,
`as well as a portion of the i-gene(i) was isolated from a
`I-Iincll digest of a pACYC 214 plasmid (a plasmid re-
`lated to pACYC 184 by insertion at a BamHI site of a
`BamHI restriction cleavage fragment from F’-lac carry-
`ing the lac gene) by repressor binding and subsequent
`
`7
`
`
`
`9
`adsorption to nitrocellose. This fragment was then em-
`ployed in the construction of an exemplary plasmid for
`analyzing strong promoters and strong terminators as
`depicted in FIG. 1.
`Utilizing the I-Ipall cleavage site within the B-gal
`structural genes the fragment was reduced in size and
`provided with Baml-ll and Sall cleavage sites by vari-
`ous subcloning. The resulting fragment (left most part
`of Figure) contains the intact control region of the lac
`operon and an N-terminal portion of the :3-gal struc-
`tural gene coding for 66 amino acids (oz). Introduction
`of this fragment by blunt end ligation into the HindIII
`site of pACYCl84 (Chang and Cohen (1978) J. Bac-
`teriol. 134,
`ll4l—ll56) yielded pBUlO. a vector suit-
`able for terminator cloning. The major terminator of the
`coliphage fd genome was isolated as a 333bp Sau3A
`fragment
`(Beck et al.
`(1978) Nucl Acids Res. 5,
`4495-4503).
`ligated with a BamHI/H.indIII adaptor
`sequence and integrated into pBUl0 to yield pLBUl.
`Cleavage of the lac sequence in pBUlO by Hpall
`destroys the lac promoter‘ and liberates upon cleavage
`with Hindlll a fragment containing the lac operator and
`a region coding for a functional tr-fragment. Integrating
`this DNA sequence into pBR322 leads to pBUl2a. Fi-
`nally,
`replacement of the Hindlll/Sall portion of
`pBU 123 by a partial digest of the I-IindIII/Sal! fragment
`of pLBU1 containing the fd terminator resulted in
`pLBU3, a vector suitable for integration of cllicient
`promoters at
`the EcoRI site. The regions encoding
`chlorarnphenicol (Cm), ampicillin (A13) and tetracycline
`{Tc} resistance are indicated as cat, bla and Let respec-
`tively.
`The plasmid. pBUlO. had the following properties: (i)
`it contains the ct fragment of B~ga.lactosida.se (tr-protein)
`and complemented the M15 deletion of the lac operon;
`{ii} the To resistance it specified was under the control
`of the lac promoter. as shown in M15 I‘? strains; (iii) the
`HindIII site between the lac gene fragment and the tel
`gene was restored; the stop codon immediately follow-
`ing the Hindlll site limited the length of the lacZ gene
`product to 63 amino acids.
`Insertion of the id terminator upstream to the let gene
`resulted in a 90% reduction in the level of Tc resistance.
`but no detectable change in the levels offi-galactosidase
`activity in M15 deletion strains. The results obtained as
`to the properties imparted to various E. coil strains by
`various plasmids is set forth in the following table:
`Seven independent plasmid isolates from clones con-
`tained the eitpected 352bp HindIII-generated fragment
`containing the Ed terminator. Electrophoretic analysis
`of all seven BamHI-cleaved isolates and DNA sequence
`analysis of one of these showed that in all instances the
`fd tenninator had been integrated in an orientation op-
`posite the direction of transcription within the fd phage
`genome. Sequence analysis also revealed a translational
`stop codon in frame with the rr-protein less than lObp
`down stream from the Hindllli cleavage forming the
`conjunction between the lac-derived segment and the
`fcl-derived DNA fragment; a translational stop signal on
`this position would be expected to result in an o.-frag-
`rncnt containing 71 amino acids.
`For the cloning of exogenous promoters. the lac pro-
`moter on the lac/tet construct had to be removed or
`destroyed in such a way that a site for the subsequent
`integration of promoters was retained. To do this. the
`Hpall cleavage site at position —l7 of lac was em-
`ployed. as described above. Colonies that showed both
`a reduced level of To resistance and the presence of a.
`
`ll]
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`4,495,280
`
`10
`lac operator sequence, which could be detected on the
`multicopy plasmid by its ability to bind the lac represser
`and induce chromosomal B-gal synthesis, were identi-
`fied. Endonuclease analysis (Hiudlll/EcoRI double
`digest} of plasmids recovered from several
`isolates
`yielded two types of vectors: one of these represented
`by pBUl2 harbored the expected 253bp lac fragment;
`the other represented by pBUl2a yielded a 42Ubp frag-
`ment. DNA sequence analysis showed that in pBUl2a,
`a 160bp fragment of unknown origin, containing two to
`three stop codons i.n each of the possible translational
`reading frames, had been integrated between the EcoRI
`site and position -15 of the lac promoter. The presence
`of the stop codons made the fragment an efficient termi-
`nator of any translation that occurred upstream of the
`translational initiation site for the cbfragment.
`When the I-Iindlll/Sail segment of pBUl2a was re-
`placed with an identically generated fragment of
`pLBUl carrying the id terminator. the plasmid pLBU3
`was obtained, which conferred neither Tc resistance
`nor B—gaI activity to E. coli M15 strain. Although this
`plasmid contained a tet region and a DNA sequence
`encoding the rz-fragment of lac, it conferred neither
`resistance nor B-gal activity to the E. colt‘ M15 strain. It
`was therefore chosen as the T5 promoter cloning vehi-
`ole.
`
`A population of about 200 short fragrnents of T5
`DNA was obtained by double digestion of the l2Dkb
`phage genome with HaeIII and Alul endonucleases.
`Thme fragments were ligated with excess synthetic
`EcoRl links and the resulting molecules were cleaved
`with Ecolll endonuclease and ligated into the Ecolll
`cleavage site of pLBU3. Transformation of E. colt‘ C600
`and selection for B-gal activity plus high level Tc-resist-
`ance yielded 35 colonies resistant to Tc-concentrations
`between 8 and 70 ug/ml. Plasmids were isolated from
`13 colonies resistant to 70 pg/ml, which earlier experi-
`ments using multicopy plasmids had suggested was the
`highest level detectable in E. colt‘ K12 (Cabello et a1.
`(1976) Nature 259, 285-290).
`Digestion of the various isolates with EcoRI endonu-
`clense liberated between one and ten fragments of vari-
`ous sizes from each constructed plasmid. Compl