WO1987000861A1 - Procede et systeme ameliores de regulation de thermochoc pour la production de produits de genes eucaryotes competents - Google Patents

Procede et systeme ameliores de regulation de thermochoc pour la production de produits de genes eucaryotes competents Download PDF

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WO1987000861A1
WO1987000861A1 PCT/EP1986/000451 EP8600451W WO8700861A1 WO 1987000861 A1 WO1987000861 A1 WO 1987000861A1 EP 8600451 W EP8600451 W EP 8600451W WO 8700861 A1 WO8700861 A1 WO 8700861A1
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gene
sequence
heat
promoter
sequences
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PCT/EP1986/000451
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Peter Bromley
Michel Dreano
Richard Voellmy
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Battelle Memorial Institute
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Priority to JP50463886A priority Critical patent/JPH01501117A/ja
Publication of WO1987000861A1 publication Critical patent/WO1987000861A1/fr
Priority to DK154187A priority patent/DK154187A/da
Priority to NO871328A priority patent/NO871328L/no

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • This technique has the intrinsic advantages as a eukaryotic expression system of having an efficient, general and highly inducible character. These factors are of considerable interest for the commercial production of proteins of pharmaceutical and other interest, particularly where these proteins are complex, modified, unstable or potentially toxic to the producer cell.
  • the natural heat-shock system in the living cell performs, amongst other roles, a complex and as yet poorly defined protective function; the nature ot heat-shock transcriptional and translational heat-shock control elements have presumably evolved in a fashion leading to an optimization of the physiological role of their associated gene products.
  • the characteristics of production and stability of heat-shock messenger RNA's and proteins are specific to their physiological roles, and are thus not necessarily those optimally desired for the heat-shock controlled production of proteins of interest. Since a number ot sequence domains involved in heat- shock promotor regions as well as heat-shock RNA leaders have been described see e.g. AMIN et al., Mol. Cell. Biol.
  • heat-shock controlling elements have also been selected from the human genome to improve compatibility between the expression system involved and the selected host-ceil machinery.
  • nucleotide insertion nucleotide deletion
  • nucleotide substitution may include single nucleotides or polynucleotide fragments.
  • the 3' and/or 5' non-translated sequences can be derived from hsp genes, foreign genes or synthetic DNA such as linkers and adapters and involve ribosome binding and translation initiation sites sequences adjacent to the ATG initiator.
  • the number of such sequences in the 5' non-translated region has been increased over that normally present in the hsp70 KDa gene; in other embodiments, the position of said sequences relative to each other and to other promoter elements has been modified.
  • the "heat shock gene control element” is defined as being composed of both a heat-shock promotor enabling control of DNA transcription into RNA, and a heat-shock RNA leader sequence influencing translational control into protein. It has generally been observed that the activity of a heat-shock promotor is independent of cell type, while the specific heat-shock translational control is largely dependent on the homology of the RNA leader and the cell or organism employed for expression.
  • the invention provides for a method tor enhancing and inducing the expression of a gene, coding for the production of a gene product (comprising a sequence of amino acids, i.e., a protein), under the control of a heat shock gene control element of the type comprising a heat shock gene translational control element, a heat shock gene promoter sequence and leader sequence, said method comprising :
  • sequences of deoxynucleotides into a fragment insertable into a plasmid and in a 5' to 3' direction comprising, in order, a heat shock gene promoter sequence, a transcription start site sequence, a 5' transcribed but not translated leader sequence for translational control in protein production, a consensus sequence coding for translation initiation, a gene sequence coding for production of a protein, and a nontranslated sequence at the 3 ' end, the heat shock gene promoter sequence comprising not more than 1500 deoxynucleotides and from 2 to 12 heat shock promoter consensus regions, each said heat shock promoter consensus region comprising not more than 11 deoxynucleotides of formula substantially approaching
  • each n is independently selected from A, T, C or G, the consensus sequence coding for translation initiation comprising not more than 20 deoxynucleotides and substantially comprising the formula
  • step 1 it is to be understood in step 1 above here and in the claims that the order is important for the DNA sequences assembled in the named order, however, additional intervening nucleotides can be Included between the ordered sequences without detriment.
  • the heat shock gene promoter is derived from the heat shock genomic DNA of Drosophilia melan ⁇ aster, particularly the genes encoding the 70 kilodalton heat shock proteins.
  • the heat shock gene control element is composed of both a heat shock promoter enabling DNA transcription into RNA and a heat shock leader sequence influencing translational control ot RNA into protein. It has generally been observed that the activity of the heat shock promoter is independent of the specific eukaryotic cell, while heat shock translation is dependent on the homology of the RNA leader and the cellular organism used for its expression.
  • the RNA leader is between the transcription start site and the translation start site.
  • the leader can be a heat shock leader enabling translation to occur at heat shock temperature or a leader from another source permitting RNA translation at normal growth temperature.
  • Fig. la represents plasmid p17, a DNA construction derived from pBR322 and incorporating a human heat-shock control sequence isolated from a human ⁇ genomic library (VOELLMY et al., PNAS USA. 82 (1985), 4949-4953).
  • Fig. lb represents plasmid pSP65 (New England Nuclear Laboratories) comprising a BamH I restriction site within a polylinker fragment
  • Fig. lc represents plasmid p17 65 JO resulting from insertion of the EcoR I (fill-in)-Hind III fragment from p17 into Pvu II-Hind III sites of pSP 65.
  • Fig. Id represents plasmid p17 JO resulting from Bgl II digestion and partial digestion with BamHI of p17 65 JO and auto ligation of a resulting 3,4 Kb fragment.
  • Fig. 2a represents plasmid phGH (obtained from Celltech Ltd., Slough, GB) containing a c-DNA fragment of a human growth hormone (hGH).
  • Fig. 2b represents plasmid pSP hGH resulting from the insertion of a BamH I fragment of phGH into the BamH I site of pSP65.
  • Fig. 2c represents plasmid p17 hGH resulting from the insertion of a Sal I - Hind III fragment of pSP hGH into corresponding restriction sites of the polylinker of p17 JO (fig. 1d).
  • Fig. 3a illustrates plasmid p17 hGH ⁇ resulting from the deletion of six nucleotides in the sequence preceding the hGH initiation of translation site.
  • Fig. 3b represents plasmid pSV2 dhfr (SUBRAMANI et al., Mol. Cell Biol. 1 (1981), 854-864) containing the mouse dihydrofolate reductase gene under the control of the SV40 early promoter sequence and terminated by the SV40 early terminator sequence.
  • Fig. 3c represents plasmid p17 hGH dhtr ⁇ 6 obtained by ligation of a 1.2 Kb EcoRI - BamH I fragment of p17 hGH ⁇ 6 into the BamH I and EcoR I sites of pSV2 dhfr.
  • Fig. 4a represents plasmid pSV2 NEO (disclosed by SOUTHERN et al., J, Mol. Appl. Genet. 1 (1982), 327-341) containing a neomycin (NEO) resistance gene under the control of the early SV40 promoter and terminated by the early SV40 terminator sequence.
  • NEO neomycin
  • Fig. 4b represents plasmid pSP NEO 9 which results from the ligation of a Pvu II - BamH I fragment derived from pSP65 and a fragment resulting from the partial digestion of pSV2 NEO with the same endonucleases (Pvu II - BamH I).
  • Fig. 4c represents plasmid p17 hGH NEO resulting from the ligation of a 1.3 Kb EcoR I fragment derived from p17hGH (see fig. 2c) with EcoR I linearized pSP NEO 9.
  • Fig. 5 is an autoradiography of an acrylamide gel electropherogram showing the various protein fractions inducihly expressed in frog oocytes using a selected gene of interest, according to the invention. Lane identification is given below; C means control temperature. Immunoprecipitation is performed with anti-hGH serum.
  • Fig. 6 refers to data similar to that illustrated in fig. 5 but genes are expressed in COS (monkey) and CHO (hamster) cells.
  • Fig. 7 is a key to DNA sequence symbols.
  • Fig. 8 illustrates the base sequences of a region of the Drosophila hsp 70KDa gene including the transcriptional and translational site and the 5' non-transcribed sequences including the promoter with the four consensus region sequences (-49 to -67, -64 to -87, -130 to -195, -191 to -430).
  • the plasmids identified in figs la to 4c include the following sequences and restriction sites (see fig. 7)
  • a plasmid, p17, derived from pBR322 and containing a functional human hs control element was obtained by screening by plaque hybridization a lambda human genomic library, using as a probe a coding sequence for the Drosophila hsp 70 KDa gene (VOELLMY et al., PNAS USA 82 (1985) 4949-4953).
  • the presence of a functional human hs control element in the DNA isolated in the cloning procedure was determined by sequence analysis as disclosed in EP-A-118 393 and the foregoing VOELLMY et al. (1985) PNAS reference.
  • a restriction fragment containing a 3.15 Kb hs non transcribed sequence and a 115 bp RNA leader was ligated with a Pvu II-Hind III fragment from pSP65 (fig 1b, a commercially available plasmid) to provide plasmid p1765 JO (tig. 1c).
  • a BamH I - Bgl II restriction fragment therefrom (3.4 Kb) containing a small 5' non transcribed sequence (0.45 kb) was autoligated to provide plasmid p17 JO (fig. Id).
  • a human growth hormone c-DNA containing clone (plasmid phGH obtained from Celltech Ltd., Slough, GB, see tig. 2a) was inserted into the polylinker of pSP65 (fig. lb) to provide plasmid pSP hGH (fig. 2b). Then a Hind III - Sal I 0,7 Kb fragment of the latter comprising the hGH sequence was inserted into the corresponding restriction site of the polylinker of p17 JO to give p17 hGH (fig. 2c); in this construction the hGH sequence is separated from the hs sequence by 27 bp including an ATG triplet before the initiation site.
  • plasmid p17 hGH ⁇ 6 contains the hGH sequence under the control of the human hs promoter, with a RNA leader sequence modified by deletion.
  • This sequence contains one for optimal translation initiation, conforming to the established consensus sequence involving strong eukaryotic translation initiation sites ( see tor instance M. KOZAK, Nature 308 (1984 ) , 241-246 ). Indeed, enhancement of the expression of the deletion modified hGH gene in suitable eukaryotic hosts compared to a control involving no deletion was observed. This will be described hereafter.
  • a 1.2 Kb restriction fragment from p17 hGH ⁇ 6 containing the hGH gene and the hs sequence was inserted into the BamH I and EcoR I sites of plasmid pSV2 dhfr (fig. 3b), the latter being obtained as disclosed by SUBRAMANI et al. Mol, Cell Biol. 1 (1981), 854-864.
  • the re suiting plasmid p17 hGH dhfr ⁇ 6 included the hGH sequence under the control of the human hs promoter ( ⁇ 6 included) and terminated by the SV40 late terminator (2) and, secondly, the dhfr gene under the control of the SV40 early promoter (1) (note the inverted reading mode).
  • the presence of the 3' late untranslated sequence also enhanced expression under stress as will be seen hereafter.
  • a plasmid, pSP NEO 9, (fig. 4b) was constructed from a BamH I-Pvu II restriction fragment of pSV2 NEO (SOUTHERN et al, J. Mol. Appl. Genet. 1 (1982), 327-341) containing the neomycin-resistance gene under the control of the SV40 early promoter and ended by the SV40 early terminator (including the poly-A addition site), and a corresponding restriction fragment from pSP 65. Digestion of p17 hGH (fig.
  • This plasmid contains two transcriptions units: first the NEO gene under the control of the SV40 early promoter and, second, the hGH gene under the control of the hs promoter with an unmodified initiation site but terminated by the SV40 early terminator. The presence of the 3' untranslated sequence also enhanced the expression of the hs controlled hGH gene in suitable host cells under stress.
  • the injected DNA-containing oocytes were subjected to stress (90 minutes at 36°C followed by incubation at 7.1°C tor 12 hours in the presence of 35 S-labelled methionine (500 ⁇ Ci/ml); the presence ot labelled protein products in either the cytoplasmic extract of the oocytes, or in the suspension media, was determined by immunoprecipitation essentially as previously described (EP-0.118.393); the results obtained are shown in fig. 5.
  • lanes M In addition to the protein molecular weight markers indicated in lanes M. two series of analyses of imraunoprecipitated proteins are shown; lanes 1-9 indicate the results obtained from the cytoplasmic extracts of the injected oocytes, while the lanes 10-18 show the corresponding results obtained from the suspension media of the same oocytes.
  • the results obtained concerning hGH synthesis are essentially identical for the two series of analyses, and the results on the secreted, extracellular proteins will be further described in the experimental part.
  • Lanes 11 and 12 show the results using plasmid p17 hGH at the control and heat-shock temperatures respectively; lanes 13 and 14, 15 and 16, 17 and 18 are comparable experiments employing plasmids p17 hGH ⁇ 6; p17 hGH dhfr ⁇ 6; and p17 hGH NEO.
  • the results may be summarized with respect to the production of a protein of molecular weight ⁇ 21 kDa specifically immunoprecipitated by the anti hGH antisera, as follows:
  • Plasmid p17 hGH which is an RNA leader fusion construct within the RNA leader sequences derived from the human hsp70 clone and lacking five nucleotides of the human sequence, produces a detectable level of hGH after heat-shock, with no apparent synthesis in the absence of heat-shock.
  • Plasmid p17 hGH ⁇ 6 is a 5' untranslated sequence modification that provides a theoretically stronger translation initiation site; this plasmid is seen to behave in a manner comparable to the previous one in the transient expression assay employed. It should be remembered that both of these plasmid constructions lack a classical 3' end terminator and poly A addition signal.
  • Plasmid p17 hGH NEO incorporates such a 3' modification by the insertion of the neomycin-resistance gene in an inverted mode with respect to the hGH sequences, and this with the unmodified initiation sequence; here a certain degree of increased efficiency of synthesis is observed.
  • p17 hGH dhfr ⁇ 6 When the two types of 3' and 5' modifications are combined in plasmid construction p17 hGH dhfr ⁇ 6, a dramatic increase in expression efficiency of hGH is observed, and this being almost totally under heat-shock control (see lanes 15 and 16, fig. 5).
  • Plasmid p17 hGH dhfr ⁇ 6 was also used to transfect cultures of COS-1 monkey cells (Gluzman, Cell 23 (1981), 175-182). The expression of human hGH in these cells was analysed using procedures essentially as described (DREANQ et al., gene (1986) in press). In this case an induction of 3 hours at 43°C followed by incubation at 37°C for a further 16 hours in the presence of 35 S-methionine was used. Control cultures that had not been heat-stressed were treated in a parallel fashion; both sets of cell cultures were then processed by immunoprecipitation with anti hGH sera and analyzed on acrylamide gels as described above. The results obtained from analyses of the suspension cultures are shown in fig. 6.
  • the next embodiments concern modifications involving the consensus sequence present in the 5' non-translated region of 70 KDa hs protein genes from Drosophila.
  • This consensus sequence of 14 bp has the formula Cnn GAAnnTTCnnG and has been suggested (PELHAM, Trends Genet 1 (1985), 31-34) between positions -49 and -62 in the 5' nontranscribed region of Drosophila heat-shock protein genes, and which is responsible for the heat-regulated expression of the genes when they are present in high numbers of copies in heterologous cell types (PELHAM Cell 30 (1982), 517 - 528; MIRAULT et al., EMBO J. 1 (1982), 1279 - 1285).
  • This sequence element is being referred to as the hs consensus sequence.
  • Region 1 extends from -49 to -67, region 2 from -64 to -87, region 3 from -130 to -195 and region 4 from -191 to -430 (see nucleotide sequence of promoter in Fig. 8).
  • a first control mutant, IN10, the promoter of which contains aforementioned consensus regions 1 and 2 only was constructed as follows: D87 DNA was digested with BssH II (cuts at -67), ends were filled in by DNA polymerase large fragment, Bgl II linkers (AGATCTAGATCT) were added, the material was double-digested with Hind III/Bgl II and Cla I/Bgl II and then electrophoresed on a 1% low melting agarose gel.
  • a 2.6 Kb Hind III/Bgl 11 fragment including region 2 of the promoter of a Drosophilia 70 KDa hsp gene and pSVOd vector sequences (MELLON et al., Cell 27, (1981) 279 - 288), including the bacterial and SV40 origins of replication and the ampicillin resistance gene and a 1.2 Kb Cla I/Bgl II fragment containing region 1 and the RNA leader region of the 70 KDa hsp gene as well as the first one third of the ⁇ -galactosidase-coding sequence were isolated.
  • a second mutant, IN20, whose promoter sequence also contains regions 1 and 2 was constructed from IN10, the DNA of which was partially digested with Bgl II (to cut the Bgl II linker between regions 1 and 2), sticky ends were filled in as before, and a Smal linker (CCCGGG) was added.
  • the sequence modification therefore involved inserting new base pairs between the two concensus sequences of IN10.
  • a hybrid test gene , R5a, which contains a promoter with one region 1 and two copies of region 2 was constructed as follows:
  • IN20 DNA was digested with Xho I, ends were filled in, the DNA subsequently digested with Hind III, and a 6.1 Kb fragment containing the hs promoter region, the ⁇ -galactosidase gene and 3' trailer sequences was isolated from a low melting agarose gel. This fragment was ligated to a 2.6 Kb Bgl II/Hind III fragment from IN10 which included promoter region 2 and upstream vector sequences.
  • hybrid genes R6a and R7a which contain promoters with one unit of region one and three or four copies of region 2 were made as follows:
  • R5a DNA was digested with Pst I and partially with Xho I. The resulting 200 and 250 bp long hs promoter fragments were isolated electrophoretically. A 1.7 kb Sma I/Pst I fragment (which included two copies of region 2 and upstream vector sequences) and a 6.9 Kb Pst I fragment (contained part of the hs RNA leader region, the ⁇ -galactosidase gene, 3' trailer and vector sequences) from R5a were isolated and ligated to the above promoter fragments.
  • mutants SE1 - 12 which only contain synthetic consensus-like sequence elements in their promoters, were assayed following a 2 hrs heat-shock at 36.5°C or following incubation of the transfected cells for the same length of time at 25 °C the control temperature. It was found that the activities of the hybrid genes are clearly a function of the number of copies of the synthetic consensus-like sequence present in their promoter regions. Significant heat-regulated activity could only be measured with mutants containing promoters with at least 3 sequence elements. Mutant SE7, containing seven sequence elements, is considerably more active in heat-shocked cells than the control gene IN20 which contains a promoter of a 70KDa hsp gene which includes regions 1 and 2.
  • a human hs 70 KDa gene was isolated as follows from a phage lambda human genomic library (LAWN et al., Cell 15 (1978), 1157-1174). The library is screened by plaque hybridization (BENTON and DAVIS, Science 196, (1977) 180-182) using as a probe a 2 Kb Sal I fragment from plasmid Sal 0 (KARCH et al., J. Mol. Biol. 148, (1981), 219-230) which contains the entire coding sequence for the Drosophila hs70 protein; any other isolate of this coding sequence can be used in place of plasmid Sal 0 as long as it contains regions of adequate practical homology to the human hsp70 gene.
  • the hs controlled hybrid plasmids were identified in transient expression experiments using microinjection into Xenopus oocyte nuclei (VOELLMY et al., PNAS. USA 79 (1982) 1776-1780).
  • One plasmid identified using such procedures as carrying human hs control sequences is plasmid p17, fig. la, see VOELLMY et al., PNAS USA (1985), 4949-4953).
  • Plasmid p17 was digested with EcoR I, filled in with DNA polymerase A Kienow fragment, and further digested with H-ind III. A 3.26 Kb fragment containing a human 3.15 Kb non transcribed sequence and an RNA leader sequence of 115 bp was purified. A Pvu II - Hind III fragment from pSP65, fig. lb (New England Laboratories) was also purified. The two fragments were ligated to give plasmid p17 65 JO. This latter plasmid was digested with Bgl II and partiallly with BamH I. A 3.4 Kb tragment was purified and "auto" ligated. This new plasmid p17 JO contains a small 5' non transcribed sequence of 0.5 kb and the above mentioned RNA leader sequence.
  • a human growth hormone c-DNA clone contained in a BamH I fragment was inserted into the BamH I site of the pSP65's polylinker to give plasmid pSP hGH.
  • Plasmid pSP hGH was then digested with Hind III and Sac 1 and the hGH sequence was inserted in the same restriction site of the polylinker of p17 JO to give p17hGH, (fig. 2c).
  • the hGH sequence was separated from hs sequences by 27 bp, and the sequence before the initiation site was... TC TAG AGG ATCC ATG G...
  • the p17 hGH was digested with Xba I and Nco I and filled in with DNA polymerase Kienow fragment, allowing a specific deletion of six nucleotides.
  • p17 hGH ⁇ 6 was digested with EcoR I and BamH I.
  • the 1.2 Kb fragment containing hGH and hs sequence was inserted by ligation into the BamH I and EcoR I sites of pSV2-dhfr (SUBRAMANI et al., Mol. Cell Biol. 1 (1981) 854-864).
  • Plasmid pSV2-NEO, 10 ⁇ g, (SOUTHERN S BERG, J. Mol. Appl. Genet, 1 (1982), 237-341) was digested with BamH I and partially with Pvu II.
  • the resulting plasmid, p17 hGH NEO contains two transcription units, first the neomycin-resistance gene (as above) and secondly, the hGH gene under the control of the human hs promoter and ended by the late SV40 terminator (including the poly A-addition site).
  • the initiation sequence is unmodified in this plasmid.
  • the scheme used for these plasmid constructions are indicated graphically in figures la to 4c.
  • the oocytes were subjected to a heat-shock of 90 minutes at 36°C followed by incubation at 21°C for 12 hours in the presence of 35 S-labelled methionine (500 ⁇ Ci/ml; 200 ul for 10-20 oocytes).
  • 35 S-labelled methionine 500 ⁇ Ci/ml; 200 ul for 10-20 oocytes.
  • the presence of labelled protein products in either the cytoplasmic extract of the oocytes, or in the suspension media was determined by immunoprecipitation of the extracts using rabbit anti hGH antisera (obtained from Dako Corporation, Santa Barbara, California; cat. no A570).
  • rabbit anti hGH antisera obtained from Dako Corporation, Santa Barbara, California; cat. no A570.
  • the detailed procedure tor immunoprecipitation and acrylamide gel electrophoretic analysis are essentially as previously described (EP- 0.118.393), the results obtained are shown in fig. 5.
  • lanes 1-9 indicate the results obtained from the cytoplasmic extracts of the injected oocytes, while the lanes 10-18 show the corresponding results obtained by immunoprecipitation of the suspension media of the same oocytes.
  • the results obtained concerning hGH synthesis are essentially identical tor the two series of analyses, and the results of the secreted, extracellular proteins will be described in detail here.
  • Lane 10 using a comparable construction to p17 hGH, but placing chicken lyosozyme gene sequence (KRIEG et al., J. Mol. Biol. 180, (1984) 615-643) under human hs control, no specific protein bands after precipitation with the anti hGH sera, even after the cells have been subjected to a heat shock as described above.
  • Lanes II and 12 show the results using plasmid p17 hGH at the control and heat-shock temperatures respectively; lanes 13 and 14, 15 and 16, 17 and 18 are comparable experiments employing plasmids p17 hGH ⁇ 6; p17 hGH dhfr ⁇ 6; and p17 hGH NEO.
  • the results may be summarized with respect to the production of a protein of molecular weight ⁇ 21 KDa, specifically immunoprecipitated by the anti hGH sera, as follows:
  • the plasmid p17 hGH which is an RNA leader fusion construct within the RNA leader sequences derived from the human hsp70 clone and lacking five nucleotides of the human sequence, produces a detectable level of hGH after heat-shock, with no apparent synthesis in the absence of heat-shock.
  • Plasmid p17 hGH ⁇ 6 is a 5' untranslated sequence modification that provides a theoretically stronger ribosome binding site; this plasmid is seen to behave in a manner comparable to the previous one in the transient expression assay employed.
  • Plasmid p17 hGH NEO incorporates such a 3' modification by the insertion of the neomycin-resistance gene in an inverted mode with respect to the hGH sequences, and this with the unmodified initiation sequence; here a certain degree of increased efficiency of synthesis is observed.
  • plasmid construction p17 hGH dhfr ⁇ 6 When the two types of 3' and 5' modifications are combined in plasmid construction p17 hGH dhfr ⁇ 6, a dramatic increase in expression efficiency of hGH is observed, and this being almost totally under heat-shock control (see lanes 15 and 16, fig. 5).
  • cytoplasmic extracts were prepared using a treatment comprising the suspension of cells in NET buffer (EP-0.118.393) followed by gentle pipetting on ice and low speed centritugation; cell suspension cultures were simply clarified by a similar centritugation step. Samples were then immunoprecipitated with anti hGH sera and analysed on acrylamide gels as described above. The results obtained from analyses ot the suspension cultures are shown in fig. 6.
  • mutants SE1 - 12 which only contain synthetic consensus-like sequence elements in their promoters, were assayed following a 2 hrs heat-shock at 36.5 °C or following incubation of the transfected ceils for the same length of time at 25 °C, the control temperature.
  • the results of these experiments are presented in Table 1.
  • hybrid genes The activities of the hybrid genes are clearly a function of the number of copies of the synthetic consensus-like sequence present in their promoter regions. Significant heat-regulated activity could only be measured with mutants containing promoters with at least 3 sequence elements. Mutant SE7, containing seven sequence elements, is considerably more active in heat-shocked cells than the control gene IN20 (this hybrid gene contains a promoter of a 70 KDa hsp gene which includes regions 1 and 2).
  • Combinations of sequence variants comprising heat-shock promotor, RNA leader, translation initiation and 3'- non-translated sequences can be assumed under certain combinations to provide optimal heat-shock expression levels of genes of interest.
  • Plasmids of interest have been deposited at the National Collections of Industrial & Marine Bacteria Ltd.

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Abstract

Dans le promoteur 70 KDa hsp, des modifications apportées au séquences 5' et 3' non-translatées, y compris l'insertion ou la délétion de nucléotides et le nombre d'éléments de séquence du concensus de thermochoc présents dans la région proximale d'un promoteur de gène de thermochoc influencent de manière critique l'activité du promoteur. Des variantes de la séquence dans l'initiation de la translation et des régions 3' non-translatées ont révélé une amélioration des niveaux de production de protéines induites. Des promoteurs mutants ayant des activités maximales supérieures à celles du promoteur parent naturel dans les cellules ayant soubi des chocs thermiques ont pu être construits. Ces promoteurs mutants sont également exprimés à des niveaux supérieurs à une température inférieure à celle requise pour l'induction optimale des gènes naturels à choc thermique. Ces résultats sont d'un intêret pratique pour des tentatives d'utilisation de promoteurs à choc thermique pour la production inductible de protéines eucaryotes d'importance médicale ou industrielle.
PCT/EP1986/000451 1985-07-31 1986-07-29 Procede et systeme ameliores de regulation de thermochoc pour la production de produits de genes eucaryotes competents WO1987000861A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP50463886A JPH01501117A (ja) 1986-07-29 1986-07-29 コンピテント真核生物遺伝子生産物の生産について改良された熱ショック制御方法および系
DK154187A DK154187A (da) 1986-07-29 1987-03-26 Forbedret varmechok kontrolmetode og system til f remstilling af kompetente eukaryotiske genprodukter
NO871328A NO871328L (no) 1985-07-31 1987-03-30 Forbedret varmesjokk-kontrollmetode og et system for fremstilling av kompetente eukariotiske geneprodukter.

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CH85810354.2(EP) 1985-07-31
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0232845A2 (fr) * 1986-02-06 1987-08-19 The General Hospital Corporation Systèmes d'amplification et de choc thermique inductibles
WO1994011521A1 (fr) * 1992-11-10 1994-05-26 Biostar Inc. Promoteur du choc thermique bovin et ses utilisations
WO2000053785A2 (fr) * 1999-03-11 2000-09-14 Glaxo Group Limited Expression
WO2005103265A1 (fr) * 2004-04-20 2005-11-03 Veterinärmedizinische Universität Wien Multiples elements de choc thermique
WO2006056825A1 (fr) * 2004-11-26 2006-06-01 Hsf Pharmaceuticals S.A. Circuits moleculaires regulateurs permettant l'activation soutenue de genes d'interet au moyen d'un stress unique
US7186698B2 (en) 1996-08-15 2007-03-06 The United States Of America As Represented By The Department Of Health And Human Services Spatial and temporal control of gene expression using a heat shock protein promoter in combination with local heat

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02500325A (ja) * 1986-08-26 1990-02-08 マッコーリー・ユニバーシティー 斑入り
ITUB20153898A1 (it) * 2015-09-25 2017-03-25 Univ Degli Studi Di Palermo Etichette intelligenti per la rilevazione visiva del deterioramento dei prodotti alimentari

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0118393A2 (fr) * 1983-02-07 1984-09-12 Battelle Memorial Institute Méthodes et compositions pour l'expression des produits des gènes compétents eucaryotes

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5447858A (en) * 1984-04-13 1995-09-05 Mycogen Plant Sciences, Inc. Heat shock promoter and gene
CA1272142A (fr) * 1984-06-11 1990-07-31 Cetus Corporation Promoteurs de mammiferes utiles dans l'expression de levures

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0118393A2 (fr) * 1983-02-07 1984-09-12 Battelle Memorial Institute Méthodes et compositions pour l'expression des produits des gènes compétents eucaryotes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Cell, Volume 35, part 1, December 1983, J.T. LIS et al.: "New Heat Schock Puffs and Betagalactosidase Activity Resulting from Transformation of Drosophila with an hsp70-lacZ Hybrid Gene", pages 403-410 see figure 1 *
Proceedings of the National Academy of Sciences USA, Volume 82, August 1985, R. VOELLMY et al.: Isolation and Functional Analysis of a Human 70.000 Dalton Heat Shock Protein Gene Segment", pages 4949-4953 see the whole article (cited in the application) *
The EMBO Journal, Volume 1, No. 11, 1982, (IRL Press Limited, Oxford, GB), H.R.B. PELHAM et al.: "A Synthetic Heat-Shock Promoter Element Confers Heat-Inducibility on the Herpes Simplex Virus Thymidine Kinase Gene", pages 1473-1477 see the whole article *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0232845A3 (fr) * 1986-02-06 1988-12-14 The General Hospital Corporation Systèmes d'amplification et de choc thermique inductibles
EP0232845A2 (fr) * 1986-02-06 1987-08-19 The General Hospital Corporation Systèmes d'amplification et de choc thermique inductibles
WO1994011521A1 (fr) * 1992-11-10 1994-05-26 Biostar Inc. Promoteur du choc thermique bovin et ses utilisations
US5521084A (en) * 1992-11-10 1996-05-28 Biostar, Inc. Bovine heat shock promoter and uses thereof
US5733745A (en) * 1992-11-10 1998-03-31 Biostar Inc. Bovine heat shock promoter and uses thereof
US5981224A (en) * 1992-11-10 1999-11-09 Biostar Inc. Bovine heat shock promoter and uses thereof
US7186698B2 (en) 1996-08-15 2007-03-06 The United States Of America As Represented By The Department Of Health And Human Services Spatial and temporal control of gene expression using a heat shock protein promoter in combination with local heat
WO2000053785A2 (fr) * 1999-03-11 2000-09-14 Glaxo Group Limited Expression
US7034142B1 (en) 1999-03-11 2006-04-25 Smithkline Beecham Corporation Method to improve translation of polypeptides by using untranslated regions from heat-shock proteins
WO2000053785A3 (fr) * 1999-03-11 2001-01-25 Glaxo Group Ltd Expression
US7351818B2 (en) 1999-03-11 2008-04-01 Smithkline Beecham Corporation Method to improve translation of polypeptides by using untranslated regions from heat-shock proteins
WO2005103265A1 (fr) * 2004-04-20 2005-11-03 Veterinärmedizinische Universität Wien Multiples elements de choc thermique
WO2006056825A1 (fr) * 2004-11-26 2006-06-01 Hsf Pharmaceuticals S.A. Circuits moleculaires regulateurs permettant l'activation soutenue de genes d'interet au moyen d'un stress unique

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AU6286086A (en) 1987-03-05
ZA865702B (en) 1987-03-25
EP0231368A1 (fr) 1987-08-12
ES2003085A6 (es) 1988-10-16
AU604214B2 (en) 1990-12-13

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