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`CONTROL NOS. 90/007,542 AND 90/007,859
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`DOCKET NOS. 22338-10230 AND -1 0231 ‘i
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`Expression of eukaryotic genes in
`E. colz
`‘
`
`-T. J. R. HARRIS
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`\
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`Celltech Ltd, 250 Bath Road,’ Slough SL1 4DY, Berks, UK
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`Introduction .
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`II Gene expression in E. coli .
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`A Transcription .
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`III Problems encountered in theexpression of eukaryotic DNA in
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`IV Expression of DNA from lovver eukaryotes.
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`V The lac promoter.
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`A The somatostatjn experiment.
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`B - Expression of insulin in E. coli .
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`Synthesis.of other hormones as B-galactosidase fusions. .
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`D Expression of ovalbumin .
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`. E Expression of native proteins . .
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`F Expression of human growth hormone .
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`VI The phage 7\ PL promoter . .
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`A Expression of eukaryotic genes from P1, plasmids .
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`VII The tip promoter . . .
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`A Construction of vectors .
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`B Expression of fusion proteins. .
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`C Expression of interferon. . .. .
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`VIII The B-lactamase promoter. .
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`A . Synthesis of fusion proteins. .p .
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`B
`Secretion of native proteins using B-lactamase fusions .
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`Synthesis of other native proteins .
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`IX Conclusions and future prospects .
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`A Alternative promoters and constructions. .
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`B mRNA structure and stability .
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`C Nature of the proteins_produced.
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`D Other host-vector systems .
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`N
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`f§i.'§"I,’.‘,‘i.E§‘.f?.'.'.“r*.‘o""""“ ‘
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`.‘;;":~!.:‘:'.".'.,‘3.’.‘l£‘;'.u".’..-12:‘;-“:.:".‘;‘ 51:1.‘
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`EVIDENCE APPENDIX
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`Genzyme Ex. 1027, pg 838
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`X Acknowledgments .
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`XI References . .
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`. 174
`. 175
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`I
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`Intro duction
`
`In recent years the techniques of in vitro DNA recombination
`followed by transfection of suitable host cells with recombinant
`vectors (gene cloning) has led to a great increase in our understanding
`of the structure and function of the genomes of many organisms. In
`the early stages of this work it became clear that genes which were
`cloned in this way could be expressed in the new host if the genetic
`elements controlling expression were suitably arranged. The results
`of these efforts will find application in two spheres. In the first, new
`approaches to fundamental studies on the relationship of protein '
`structure to function will be possible. Already, molecules have been
`produced which are hybrids of the appropriate regions of different
`interferon molecules and their functions are being examined. This is
`possible not only because the genes for the proteins can be recom-
`bined but because they can then be expressed in E. coli in quantities
`sufficient for purification and biological study (Streuli et al., 1981;
`Week et al., 1981). Further extensions of this kind of work can be
`foreseen where one or a few selected amino acids (e.g. near the active
`site of an enzyme) are altered by in vitro mutagenesis (Shortle et al.,
`1981; Lathe et al., 1983) and the effect on enzymatic function
`assayed. Secondly, such is the power of these gene cloning and
`expression techniques that
`they are already having a profound
`impact on the practice of biotechnology and it seems that few areas
`of this technology will remain unaffected by them. Indeed, the first
`proteins made by recombinant DNA techniques are now being pro-
`duced in sufficient quantity for extensive safety and efficacy testing.
`Insulin and growth hormone, both conventionally isolated from
`human endocrine tissue have now been made in E. coli and the
`
`proteins purified (Goeddel et al., 1979a, 1979b). Considerable effort
`has been expended on the isolation and expression of both leukocyte
`(Le or oz) and fibroblast (F or B) interferon genes so that the potential
`of these antiviral compounds can be evaluated properly ‘(see Scott
`and Tyrrell, 1980). There is also the possibility of producing proteins
`for use as vaccines against a variety of infectious agents by cloning
`and expressing the genes coding for the relevant surface immunogens.
`Notable progress has been made towards a vaccine for foot and
`mouth disease virus (FMDV) using this approach, where one of the
`capsid proteins (VPI) produced in E. ‘coli has been shown to elicit
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`EVIDENCE APPENDIX —
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`Genzyme Ex. 1027, pg 839 '
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`Expression of eukaryo tic genes
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`129
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`neutralizing antibody (Kleid et al., 1981). Genetically engineered
`vaccines for other viruses such as hepatitis B and rabies virus are also
`being considered.
`.
`-
`Although none of these initial examples. of the expression of
`proteins from recombinant organisms is as yet established as a bio-
`technological process, the way in- which the expression of the recom-
`binant DNA was achieved forms a general paradigm for all future
`studies. However, at the same time, it is clear that not all the rules
`governing ‘the expression of cloned genes have been elaborated and
`those rules that do exist are still largely empirical. In this article
`the ways in which expression has been achieved are reviewed, some
`of the problems discussed and some of the probable future systems
`considered.
`
`II Gene expression in_E. coli
`
`E. coli has been used as the host-cell for expression of foreign genes
`mainly because more is known about the control of gene expression
`in this organism than in any other. It is well established, for example,
`that the genes involved in a particular metabolic activity tend to be
`clustered in transcriptional units (operons) with the major control
`regions (the operator and promoter) located at the beginning of the -
`cluster (for a detailed description of bacterial gene expression, see
`Miller and Reznikoff, 1980). The operon is transcribed into a poly-
`cistronic mRNA from which the polypeptides are then translated.
`Transcriptional control is exerted over the expression of an operon
`and varies depending on the function of the genes in the operon
`(see Miller and Reznikoff, ‘1980). Since relatively few promoter
`systems are currently being utilized to express cloned‘ genes, the
`essential elements, of their control mechanisms will be dealt with
`when considering each system. Expression of a cloned gene requires
`efficient and specific transcription of the DNA, translation of the
`mRNA and in some cases post-translational modification of the
`resulting protein .
`I
`'
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`V Transcription
`
`The first step in the initiation of transcription in E. coli is the binding 4
`of RNA polymerase to a promoter sequence in the DNA. Analysis of
`the DNA sequence of many promoters in E. coli has revealed two
`regions of homology located "about 35 base pairs (bp) upstream
`from the transcription initiation site (the - 35 region) and about
`10 bp- upstream (the -10 region or Pribnow-Schaller box). The
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`EVIDENCE APPENDIX
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`T. J. R. Harris
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`conserved sequences in the - 35 and - 10 regions (TTGACA and
`TATAAT respectively, Rosenberg and Court, 1979; Siebenlist et al.,
`1980) probably represent those bases most intimately involved in
`polymerase binding and orientation via sigma factor, so that RNA
`chain initiation can take place just downstream.
`Transcription termination is also controlled by signals in the DNA
`sequence, characteristically a GC rich region having a two-fold
`symmetry before the termination site, followed by an AT rich -
`sequence at the site of termination (Rosenberg and Court, 1979).
`Several protein factors are also involved in ‘the control of term-
`ination, most notably the rho factor. Anti-termination proteins such
`as the N gene product ofephage 7\ can also be involved in specialised
`systems (Greenblatt et al., 1981).
`
`B Translation
`
`Efficient translation of mRNA in prokaryotic cells requires the
`presence of a ribosome binding site (rbs). For most E. coli mRNAs-
`the rbs consists of two components, the initiationcodon AUG and,
`lying 3--12 bases upstream, a sequence of 3-9 bases called the
`Shine-Dalgamo .(SD) sequence complementary to the 3' end of the
`16S rRNA (Shine and Dalgarno, 1975). It is ‘believed that hybrid-
`ization to this region is involved in the attachment of the ribosomal
`30S subunit to the mRNA (Steitz, 1979). The SD sequence is not
`identical in all mRNAs but a semi-conserved consensus sequence has
`been identified just as for promoter sequences. It is possible that
`differences in SD sequences form part of a translational control
`system. In addition, ribosome binding is probably modulated by the
`secondary structure at the 5' end of the RNA since more efficient
`translation occurs if the AUG and SD sequence are freely accessible
`to 30S ribosomal subunits (Iserentant and Fiers, 1980). Termination
`of translation usually occurs whenever one of the three stop codons
`is encountered in the mRNA by a ribosome complex, provided that
`an.aminoacylated suppressor tRNA is not present.
`
`0 Post-translational modification
`
`There are a variety of modifications that bacterial proteins can
`undergo following transl'ation.T-he forrnyl group on the NH,-terminal
`methionine is hydrolysed and one or more NH,-terminal residues
`may be removed. Many secreted proteins are synthesized as large
`precursors with
`additional
`hydrophobic NH,-terminal
`signal
`sequences _that are cleaved off by a membrane bound enzyme (for
`~ review,
`see Davis and Tai, 1980). However, glycosylation and
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`phosphorylation, which are common modifications of proteins in
`eukaryotic cells do not occur to any great extent in E. coli.
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`Expression of eukaryotic genes
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`131
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`IH " Problems encountered in the expression of
`eukaryotic DNA in E. coli
`
`Successful expression of a eukaryotic gene in E. coli requires that the
`cellular machinery is organised so that the level of expression of
`‘ the cloned gene is as good or better than the resident genes; Probably
`the most important difference between eukaryotic genes (at least
`from higher organisms) and prokaryotic genes is the presence of
`intervening sequences (introns) which interrupt the coding sequences.
`Normally these sequences are spliced out of the initial RNA
`transcript, producing cytoplasmic mRNA suitable for translation.
`There are no introns in prokaryotic genes and consequently no
`splicing enzymes present, so in general genomic DNA ‘cannot be used
`as a source of genes. for expression in bacterial cells. A second
`problem is that transcriptional signals in eukaryotes are different
`from those in prokaryotes (Corden et al., 1980; Breathnach and
`Chambon, 1981) -and are not usually recognised by bacterial RNA
`polymerase. This difference again emphasizes the fact that eukaryotic
`genomic DNA is not a suitable gene source for construction of
`expression vectors. Thirdly, the structure of eukaryotic mRNA is
`different to bacterial mRNA. Eukaryotic mRNA is polyadenylated '
`at the 3' end and normally capped at the 5’ end, features which
`may affect mRNA stability and ribosome binding (Breathnach and
`Chambon, 1981). Furthermore eukaryotic mRNA does not seem to
`have an equivalent of the SD sequence present in prokaryotic mRNA
`(Kozak, 1981).
`_
`—
`An additional problem is that of codon usage. The codons used
`in mRNA coding for highly expressed prokaryotic genes are not
`random;
`there is a marked preference for particular codons for
`some amino acids (Grantham et al.,' 1981; see Grosjean and Fiers,
`1982). This preference appears to correlate with the abundance of
`different tRNA species (Ikemura, 1981). As codon selection pref-
`erences are different for eukaryotic genes it is possible that the
`levels of certain tRNAs will affecttranslational efficiency of these
`genes in a prokaryotic system. Finally,
`it
`is known that many
`eukaryotic proteins are subject to '_a number of post-translational
`modifications which may affect either activity or stability. Most of
`these modifications do not occur in prokaryotes.
`‘
`-
`A number of strategies have been developed_to try to overcome
`these difficulties (Table 1). Onceithe amino acid sequence of a
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`EV|DENC.E APPENDIX
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`T. J. R. Harris
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`Table 1
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`General strategies for the expression of cloned genes in E. coli.
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`Control level
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`Strategy
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`Gene
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`Transcription
`"(Initiation and termination)
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`Translation
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`Synthesise DNA in uitro by chemical methods,
`with optimised codon assignments or obtain
`cDNA clone to specific mRNA. Chemical DNA
`synthesis probably required for tailoring genes
`into expression vector.
`
`Clone gene adjacent to strong E. coli promoter
`which is controllable so that transcription can
`be induced (derepressed) when required. Use a
`multicopy plasmid to increase gene dosage.
`Include termination signal after gene to prevent
`transcriptional read-through.
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`Fuse gene in correct translational reading frame
`to an E. coli gene already in the vector, so that
`normal rbs is maintained. Possible to use both
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`long and short NH,-terminal fusions.
`Alternatively, place new gene with its own
`AUG adjacent to an rbs. The sequence of the
`SD sequence and distance from the initiating '
`AUG may modulate translation. Accessibility
`(secondary structure) around SD-AUG may be
`important. Codon usage can be overcome by
`using chemically synthesized genes. Not clear
`if codon bias actually affects the translation
`-of cloned genes. Include stop codon (s) in
`chemically synthesised genes.
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`'
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`Protein
`(Secretion and stability)
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`Use signal sequences to control secretion?
`Synthesis of precursor proteins followed
`by their processing ensures removal of
`NI-I2-terminal initiating methionine. Factors
`affecting folding of foreign proteins and their
`degradation in E. coli are not well defined.
`Synthesis of long fusion proteins may result in
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`increased stability. '
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`protein is known it is now a relatively straightforward task to design
`and synthesize chemically, a DNA sequence that will code for the
`protein without
`the problem of intervening sequences and with
`optimized codon assignments. A gene of 514 bp coding for leukocy.te
`Le (oz) interferon, a protein of 166 amino acids is the longest DNA
`sequence that has been synthesized so far
`(Edge et al., 1981).
`"Although there is no theoretical limit‘ to the size of gene that can be
`synthesized, practical problems arise for much larger proteins. If the
`gene is too big for a chemical synthesis, then double stranded DNA
`copies of mRN A populations can be generated, cloned into a plasmid
`vector and the clone containing the sequence coding for the _protein
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`EVIDENCE APPENDIX
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`Expression of eukaryotic genes
`133
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`J,
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`of interest selected from the clone bank by hybridization tech-
`niques.
`-
`‘ Transcription_ of these genes is controlled by inserting the DNA
`adjacent to a strong prokaryotic promoter in a cloning vector. Four
`promoters have been used most widely for this purpose, the lac
`promoter from the E. coli ,lac operon;. the trp promoter from the
`E. coli trp operon; the strong leftward promoter of phage 7\ (PL) and
`the constitutive and weaker (3-lactamase promoter present in the
`plasmid vector pBR322. The expression vectors themselves are
`usually derived from high copy number plasmids so that" there is
`increased expression owing to gene dosage (Gelfand et al., 1978;
`O’Farrell et al., 1978). Termination of transcription can be ensured
`by placing a termination site after the cloned gene (e.g. Nakamura
`and Inouye, 1982) although whether this is necessary for the main-
`tenance of high levels of transcription isnot yet clear. The conse-
`quences of uninterrupted transcription around a small circular
`plasmid DNA molecule are -unknown. It is presumably detrimental
`since most expression vectors’ have other genes present (e.g. an
`antibiotic resistance gene) which are transcribed in the opposite
`direction from a different promoter and it isiknown that the trans-
`‘cription of genes in A phage carrying the trp promoter is adversely
`affected if the trp promoter is in an orientation where transcription
`occurs towards transcripts arising from the PL promoter (Hopkins
`et al., 1976).
`'
`Translational barriers have been overcome to some extent by two
`procedures. The foreign gene is ‘either fused (in the correct trans-
`lational reading frame) to a prokaryotic gene so that the existing rbs
`is‘ used to initiate translation, or the new gene, with its own initiation
`codon, is placed adjacent to a naturally occurring E. coli rbs (Backman
`et al., 1980) or asynthetic one (Jay et al., 1981). Since all structural
`genes, whether eukaryotic or prokaryotic, end with one or more of
`the three termination" codons it is not usually necessary to make
`special arrangements for translational
`termination when using a
`cloned cDN.A sequence. However, _ a termination codon must be
`included when synthetic DNA is used.
`Protein modification and stability are much less easy to control,
`largely because the structural features governing protein stability in
`E. coli are not well understood. It has been shown that eukaryotic
`signal sequences are recognised by'E. coli and that NH,-tenninal
`fusions of eukaryotic polypeptides to E. coli signal sequences results
`in secretion of the protein to the periplasmic space, with concomitant
`cleavage of the signal sequence (Talmadge et az., 1980;,1981). There
`is also some evidence that short “foreign” polypeptides are unstable
`in E. coli (Itakura et al., 1977; Goeddel et al., 1979a). This has been
`
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`overcome by fusing the peptide to a larger E. coli protein from which
`the peptide is then cleaved.
`
`IV Expression of DNA from lower eukaryotes
`
`Following the observation that DNA from S. aureus could be
`expressed in E. coli (Chang and Cohen, 1974) it was shown that
`eukaryotic DNA could also be transcribed (Morrow et al., 1974;
`Chang et al., 1975; Kedes et al., 1975). It was not clear from these
`experiments, however, whether the normal transcriptional start and
`stop signals were being recognised. The fundamental question of
`whether a fungal gene could be transcribed and translated to produce
`a functional protein in E. coli was answered to some extent by the
`finding that fragments of yeast DNA cloned into phage 7x, or the"
`plasmid vector Col E1 could _complement auxotrophic mutants of
`E. coli (e.g. His B and Leu B) (Struhl et al., 1976; Ratzkin and
`Carbon, 1977; Struhl and Davis, 1977). Similarly segments of Neuro-
`spora crassa DNA containing the gene for dehydroquinase were
`‘ successfully expressed in E. coli in a pBR322 replicon (Vapnek et al.,
`1977 )§ Several other yeast genes have now been expressed in this way
`'(e.g. Trp 1, Trp 5 and Arg 4). The functional expression of yeast
`DNA in E. coli not only demonstrated that eukaryotic DNA could be
`transcribed and _t3anslate_d, paving the way for the experiments
`described below, but.also provided a powerful method for isolating
`yeast genes. Some of these genes have subsequently been used to
`provide selection markers in yeast—E. coli shuttle vectors (Beggs,
`1982; Hinnen and Meyhack, 1982).
`
`'
`
`The lac promoter
`
`The lac operon is subject to two types of control. In the absence of
`lactose (or other inducer) the operon is kept switched off by lac
`repressor (the lac i gene product) binding to the operator. Positive
`regulation is also exerted through the catabolite gene activator
`protein (CAP). In the absence of glucose, CAP forms a complex with
`cyclic AMP and this complex stimulates transcription by binding
`next to the promoter. The operon is derepressed by the_ presence of
`lactose,.or by the addition of the non-metabolizable inducer IPTG
`(isopropylthiogalactoside) which binds to the repressor and removes
`it from the operator.
`~
`:
`
`EVIDENCE APPENDIX
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`135
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`I
`
`Plasmid vectors containing parts of the lac operon have been
`constructed by several workers. Polisky et al. (1976) cloned an
`EcoRI fragment from A p lac 5 DNA (a transducing phage containing
`part of the lac operon) into a C01 E1-derived plasmid to obtain
`a vector with the lac promoter and operator and most of the B-
`galactosidase. gene. Plasmids containing a small “portable” lac
`promoter fragment have also been made. In these constructions a
`203 bp HaeHI fragment -of lac transducing phage DNA, containing the
`lac promoter and operator and first eight codons of B-galactosidase,
`was blunt end ligated into EcoRI-cut and “filled in” pBR322 DNA.
`The portability derives from the fact that EcoRI sites are reformed
`at the junctions allowing the promoter fragment to be removed by
`EcoRI» digestion (Backman and Ptashne, 1976; Itakura et al., 1977).
`Colonies harbouring plasmids which carried the lac ‘promoter-operator
`were identified by their constitutive synthesis of B-galactosidase,
`rendering’ them blue on agar plates containing X gal (5 chloro-4
`' bromo 3 indolyl-D galactoside). This is because multiple copies of
`the operator fitrate out all the lac repressor resulting in derepression
`of the chromosomal B-galactosidase gene. Both A p lac 5 DNA and_
`7x h8O lac UV5 C1857 DNA, which contains the CAP site mutation
`L8 and the up promoter mutation UV5 (making the promoter insen-
`sitive to catabolite repression), have been used as a source of lac
`DNA for these constructions (Backman et al., 1976; Itakura et al.,
`1977; see also Fuller, 1982). Further derivative plasmids containing a
`95 bp Alul fragment of lac DNA,including the UV5 promoter (minus
`the CAP binding site), the repressor binding site and most of the rbs,
`just excluding the ATG of [3-galactosidase, have also been con-
`structed for the expression of non-fusion proteins (Fuller,-1982).
`
`A The somatostatin experiment
`
`The first report of the designed expression of a eukaryotic gene in
`E. coli was the production of the small peptide hormone somato-
`statin (Itakura et al., 1977). Somatostatin was used as a model
`‘system because the hormone was a small polypeptide of known A
`amino acid sequence for which sensitive radioimmune and biological .
`assays existed. The experiments illustrate a number of features of
`methods which are now used to "obtain expression of cloned genes.
`They ‘also demonstrated, although not for the first
`time,
`that
`_ chemically synthesized DNA was functional in a biological system.
`In addition, the production of the protein as a fusion polypeptide
`and its subsequent cleavage into the native hormone at methionine
`residues by cyanogen bromide (CNBr), has been used quite extensively"
`. for other proteins. This overall strategy is depicted in Fig. 1.
`
`'
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`EVIDENCE APPENDIX
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`PAGE B475
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`Genzyme Ex. 1027, pg 846
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`Genzyme Ex. 1027, pg 846
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`T. J. R. Harris
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`E. coli Lac Operon DNA
`
`chemicm
`
`GENETIC CODE
`
`-—
`
`‘
`
`.
`
`1
`
`DNA Synthesis
`Somotostahn Gene
`[5-Gal
`P 0
`Lac
`..—j.j_.:_.j._..j... 357 gm’ 751' gm; AA‘; ffc 711' 1-6
`AA G
`pBR322
`Plasmid om:
`1“
`1 In Vivo
`
`GAT AGT TGT GCT YCA CTT TCA (3
`
`Sam
`15- Gal
`NH2 Mev - Mo‘ Glv - Cvv Ly:-Asn - Phe- Pr-2.
`é
`§
`HO - Cy8-Ser-Thr- Phe -Thr'
`
`Tip
`Ly!
`
`in Vitro
`
`Cyanogen Bromide
`Cleavage
`
`'
`
`-Gal Fro ments
`9
`
`+
`
`NH - Ala - Gly - cys- Ly;-am - PM - PM.
`2
`|
`T’
`s
`‘
`.Lv-
`i’
`HO - Cyt- Ser-The - Phe -.TM ~
`
`T
`
`Active Somotoslatin
`
`Strategy for the expression of the chemically synthesized somato-
`Figure 1
`statin gene as a ll-galactosidase fusion from the lac promoter. The active hormone
`can be cleaved from the hybrid protein by CNBr treatment. (Reproduced from
`Itakura et al., 1977, copyright by the American Association for the Advancement
`of Science, with permission.)
`'
`
`In the first set of experiments the chemically synthesized somato-
`statin gene with synthetic EcoRI and Bam HI cohesive ends" was
`cloned into a vector containing the wild type Hae III lac promoter
`fragment. The DNA sequence indicated thatthe plasmid should have
`produced a polypeptide containing the first seven amino acids of
`' B-galactosidase fused to somatostatin. However, no somatostatin was
`detected in bacterial extracts by radioirnmunoassay. As. it was found '
`that somatostatin was not‘ stable when added to E. coli extracts, the
`failure to find somatostatin was thought to be due to proteolytic
`digestion (Itakura et al., 1977). The approach adopted to try to
`stabilise the somatostatin was to produce it as part of a longer
`polypeptide from which it could be cleaved by CNBr. This was done
`by linking the somatostatin gene to the EcoRI fragment of A p lac .
`5 DNA which carries the lac promoter and a large proportion of the
`B-galactosidase gene (-Polisky et al., 1976). The translation reading
`
`EVIDENCE APPENDIX
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`PAGE B476
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`Genzyme Ex. 1027, pg 847
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`Expression of eukaryotic genes
`frame of B-galactosidase was maintained in somatostatin after fusion
`_at__the_:Eco_BI_ junction. In these constructions only one orientation
`of the EcoRI lc_zc fragment maintained the correct reading frame in
`so‘matostatin" and _when several independent clones were examined,
`about half produced detectable somatostatin after CNBI‘ cleavage.
`No immunoreactive protein was detected before cleavage since the
`antiserum used in the assay required a free NH,-terminal alanine
`residue (Itakura et al., 1977).
`
`.
`
`\
`
`'
`
`.
`
`B Expression of insulin in E. coli
`
`The somatostatin work established the feasibility of the synthetic
`gene fusion approach for the expression of small polypeptides in
`E. coli. It was possible to follow an almost identical strategy to
`obtain expression of human -insulin, as neither the 20 amino acid
`A chain nor
`the 30 amino acid B chain "of
`insulin contained
`
`methionine and methods were available for the in vitro joining of the
`two chains. Thus, an A chain gene and a B chain gene were chem-
`ically synthesized each with Bam HI and EcoRI cohesive ends (Crea
`et al., 1978) and cloned separately into pBR322. The B chain gene
`was synthesized with a Hindlll site in the middle so that the two
`halves could be cloned separately and the sequence verified (Goeddel
`et al., 1979a). Expression was achieved by transcription from the
`same lac promoter as used for the successful somatostatin con-
`structions and insulin A or B-[3-galactosidase fusion proteins were
`produced (Goeddel et al., 1979a). The hybrid proteins represented
`‘about 20% of total cell protein, which was about ten-fold higher than
`the level of expression obtained with somatostatin. The hybrid
`proteins were insoluble and were found in the first low speed pellet
`after breaking the cells with a French press.
`To obtain A and B peptides suitable for reconstitution into
`native insulin,
`the hybrid proteins had to be solubflised, the B-
`galactosidase portion removed and the peptides S-sulphonated. This
`was achieved by dissolving the hybrid proteins in 6 M guanidinium
`chloride followed "by dialysis. The precipitate was dissolved in 70%
`formic acid,
`the protein cleaved with CNBr and S-sulphonated
`_ derivatives of the peptide mixture obtained, using sodium dithionate
`and sodium sulphite at pH 9. Insulin activity was readily detected
`by radioimmunoassay after re-constitution. Further studies on the
`‘ peptides (e.g. chromatographic behaviour) and amino acid com-
`positions established, without doubt, that the bacteria were pro-
`ducing authentic insulin A and B chains (Goeddel et al., 1979a).
`Insulin, prepared from bacteria containing these constructions by a
`scaled up and modified process, has now been shown to be active
`
`EVIDENCE. APPENDIX
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`PAGE B477
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`Genzyme Ex. 1027, pg 848
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`Genzyme Ex. 1027, pg 848
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`CCNTROL NOS. 90/307,542" AND ‘.90/007,859 '
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`T. J. R. Harris
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`DOCKET NIOS. 22338-10230 AND -10231
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`when injected into human volunteers (Clark et al., 1982) and to
`interact with insulin receptors in the same way as native human
`insulin (Keefer et al., 1981).
`An alternative approach involves the synthesis of a gene coding for
`proinsulin, the natural precursor to insulin. Proinsulin is synthesized
`initially as a preproinsulin molecule consisting of an NH,-terminal
`signal sequence, followed by the B chain, a linking C peptide and the
`COOH-terminal A chain. Enzymatic removal of the signal peptide
`during secretion generates proinsulin and processing at two _trypsin
`sensitive sites (Arg-Arg, Lys-Arg) allows the removal of the C peptide
`and the generation of active
`The three dimensional structure
`of insulin indicates that a peptide much shorter than the 35 amino
`acid connecting C pepfide should be sufficient to connect the B and
`A chains and still allow proper folding of the modified proinsulin.
`Genes coding for human proinsulin and “mini C" derivatives of
`proinsulin, where the C peptide is replaced by a six amino acid linker
`retaining the proteolytic cleavage sites, have been constructed by
`chemical synthesis (Sung et al., 1979; Wetzel et al., 1981a; Brousseau
`et al., 1982).
`'
`l
`The mini C construction . was cloned for expression as a [3-
`galactosidase fusion protein (Wetzel et al., 1981a) and a product with
`a-proinsulin~like structure (as determined by radioimmunoassay and ‘
`HPLC) was detected after CNBr cleavage and S-sulphonation. The
`usefulness of this route to insulin production is still not clear
`however, as there are no data on the behaviour of mini C derivatives
`in enzymatic» proinsulin processing systems and there are already
`preproinsulin expression constructions available derived from cDNA
`' (see B-lactamase section). However, the modular approach to the
`chemical synthesis of proinsulin adopted by Brousseau et al. (1982)
`does have the advantage that the shortening and changing of parts
`of the C peptide or alteration of the codons can be approached
`rationally by the incorporation of different oligonucleotide blocks
`during synthesis, obviating the need to synthesise an entire coding
`sequence each time a specific modification is made.
`
`C Synthesis of other hormones as B-galactosidase fusions '
`
`The strategy of using the lac promoter/operator and [3—galactosidase
`NH,-terminal. fusions has been adopted for several other proteins
`including other hormones (see Table 2). For example the neuro-
`peptide [3-endorphin, a 30 amino acid endogenous opioid has been
`expressed in this way (Shine et al., 1980). In these experiments a
`cDNA clone to the precursor peptide of mouse corticotropin (ACTH)
`and [3-lipotropin -(LPH). w