WO2003100047A1 - Clonage, expression et caracterisation de la sequence complete d'une phosphodiesterase 8b - Google Patents

Clonage, expression et caracterisation de la sequence complete d'une phosphodiesterase 8b Download PDF

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Publication number
WO2003100047A1
WO2003100047A1 PCT/EP2003/005429 EP0305429W WO03100047A1 WO 2003100047 A1 WO2003100047 A1 WO 2003100047A1 EP 0305429 W EP0305429 W EP 0305429W WO 03100047 A1 WO03100047 A1 WO 03100047A1
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Prior art keywords
sequence
pde8b
protein
nucleic acid
expression
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PCT/EP2003/005429
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German (de)
English (en)
Inventor
Thorsten Hage
Ute Egerland
Christin Galetzka
Dietrich Dettmer
Thomas Hermsdorf
Liviu Unciuleac
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Elbion Ag
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Application filed by Elbion Ag filed Critical Elbion Ag
Priority to AU2003240701A priority Critical patent/AU2003240701A1/en
Publication of WO2003100047A1 publication Critical patent/WO2003100047A1/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • the present invention relates to the overall sequence of phosphodiesterase 8B (PDE8B) from the thyroid.
  • PDE8B phosphodiesterase 8B
  • the third methionine residue at amino acid position 79 in the reading frame fulfills the conditions of a Kozak consensus sequence for the start of translation in eukaryotes, which is why an N-terminally truncated but nevertheless enzymatically active protein of 584 amino acids corresponds to amino acid positions 76 - 659 and base positions 226 - 1 977 was expressed.
  • the N-terminally truncated protein has a molecular weight of 66 kDa.
  • Phosphodiesterase 8B specifically cleaves cAMP and is only inhibited by dipyridamole, but not by rolipram, IMBX, vinpocetine, cGMP and milrinone. A particularly high expression of the enzyme is found in the thyroid.
  • the object of the present invention was to provide the complete coding sequence for PDE8B and thus to enable the expression of a complete protein.
  • the object was achieved by providing the complete DNA sequence which codes for human PDE8B.
  • the new complete sequence according to the invention is shown in Figure 1 0.
  • the protein with the correct size could be detected in a Western blot with PDE8B-specific antipeptide antibodies.
  • the PDE8B gene is located on chromosome 5q 1 3.3. Alignment of the sequence presented here with genomic DNA using the blastp program was only partially successful. A good agreement could be achieved within the 3 'sequence region, whereas no results were obtained at the 5' end and in the middle. An analysis without such a program led to the assumption that the sequence coding for PDE8B in the genome should consist of 29 exons. In the middle of the sequence there is a series of very short exons. There is also an intron greater than 1 33 kbp between the first and second exons. Finding the complete PDE8B sequence was therefore extremely difficult.
  • the DNA sequence according to the invention makes it possible for the first time to recombinantly produce the intact and complete human PDE8B. Another subject of the present application is therefore a recombinantly produced PDE8B.
  • expression vectors which the DNA sequence according to the invention included and can therefore be used to express the complete PDE8B.
  • a particularly preferred vector containing the entire sequence for PDE8B is the vector pCMV5, which was produced in accordance with Example 3.
  • Host cells which are transfected with a corresponding expression vector are also a further subject of the present invention.
  • the cultivation and extraction of PDE8B from these host cells is carried out according to methods known to the person skilled in the art.
  • the protein produced according to the invention can i.a. used to test substances for their ability to influence PDE8B. In particular, the investigation of substances for their ability to inhibit PDE8B is of particular interest.
  • sequence and structural information can be obtained from the recombinantly produced protein according to the invention, which make it possible to find sub-areas of the protein which are essential for the effectiveness of the protein. In this way, the necessary basics for the production and design of active ingredients can be provided. Active ingredients can both reduce or block enzyme activity or also have a positive effect.
  • the provision of the complete PDE8B protein also makes it possible for the first time to study the interactions with cellular components in detail.
  • PDE inhibitors have already been tested using the enzyme according to the invention.
  • the substance EHNA and dipyridamole showed the greatest effect on the inhibition of PDE8B at a concentration of 100 ⁇ M (see Fig. 1 1).
  • Another subject of the present The invention is therefore the use of the complete PDE8B enzyme according to the invention for the investigation of substances for their ability to modulate, in particular to reduce or inhibit the activity of the enzyme.
  • substances can then be of particular interest in connection with the treatment of thyroid disorders.
  • these substances can also be used in connection with diseases of other organs in which the PDE8B plays a role.
  • Reverse transcriptase was used to synthesize cDNA from total thyroid gland (Stratagene).
  • the cDNA served as a template for the PCR.
  • the PDE8B type-specific primer was designed using the Oligo, Medporbe program.
  • a suitable upper primer was only found in base position 1 2. No amplificate could be obtained with the primer combination 1 2 upper and 2030 lower (Fig. 1). Further primer combinations were therefore tested.
  • the primer combination 249 upper - 2030 lower resulted in the longest fragment (Fig. 1). With the primer combination 1 2 upper and 691 lower, the majority of the remaining 5 'sequence was shown separately (Fig. 1).
  • the 5'-RACE was performed using the methodology of Maruyama, I.N. et al., Nucleic Acids Research (1 995) 23, 3736-3797, which has been slightly modified for our application. This method is also the basis of Takara's 5'-RACE kit. Reverse transcriptase was used to synthesize 691 cDNA from total thyroid gland (Stratagene) and the 5'-phosphorylated lower primer. The cDNA single strands were ligated with T4-RNA ligase and the ligation product was used as a PCR template. A primer combination (532 upper / 351 lower) was selected as the PCR primer, which ensures that the ligation site is in the amplificate. The methodology is shown schematically in Fig. 2.
  • An amplificate of approximately 1000 bp could be obtained (Fig. 3).
  • the primer combination 532 upper / 351 lower results in an amplificate via the ligation site of at least 498 bp.
  • a sequence extension of approximately 500 bp could therefore be achieved.
  • the 1000 bp amplificate was cloned into the vector pCRII-TOPO (Invitrogen) by TA cloning (FIG. 4) and then sequenced.
  • the results show a sequence extension in the reading frame based on the known sequence with a start codon in the ORF (Fig. 5).
  • the start codon fulfills all requirements of a Kozak consensus sequence for the start of translation in eukaryotes. It can therefore be concluded that the complete 5 'end of the PDE8B could be shown.
  • the functionality of the pCMV5 vector with the PDE8B insert was tested by transient transfection of COS cells.
  • the first cytosol was able to detect fiftyfold overexpression in the first orientation tests (Fig. 1 1 and Fig. 12).
  • the K M value is 0.5 ⁇ M.
  • the Bac to bac system (Invitrogen) was used for expression of the new PDE8B protein in Sf9 insect cells.
  • the new, previously unknown "fill length" PDE8B sequence was cloned into a pFASTBACI vector using the method described above. Protein expression was analyzed by Western blot (Fig. 1 2). No PDE activity could be determined in the fractions of membrane and cytosol in the non-transfected cells. In comparison, an overexpression of the PDE8B protein of at least 400-fold was detected in the transfected Sf9 cells (Fig. 1 1). The determined K M value for the cytosol fraction is 0.5 ⁇ M.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un acide nucléique codant une PDE8B, qui comprend (a) une séquence d'ADN représentée sur la figure 10, (b) une séquence différente de ladite séquence et codant cependant la même séquence d'acides aminés, (c) une séquence codant une protéine présentant des mutations ou des délétions qui ne modifient pas sensiblement l'activité ou (d) une séquence s'hybridant à la séquence représentée sur la figure 10, qui code une protéine présentant une activité de PDE8B. Un vecteur d'expression selon cette invention renferme une telle séquence qui est insérée dans un vecteur adapté.
PCT/EP2003/005429 2002-05-23 2003-05-23 Clonage, expression et caracterisation de la sequence complete d'une phosphodiesterase 8b WO2003100047A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003240701A AU2003240701A1 (en) 2002-05-23 2003-05-23 Cloning, expression and characterisation of the total sequence of phosphodiesterase 8b

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2002122845 DE10222845A1 (de) 2002-05-23 2002-05-23 Klonierung, Expression und Charakterisierung der Gesamtsequenz von Phosphodiesterase 8B
DE10222845.0 2002-05-23

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WO2003100047A1 true WO2003100047A1 (fr) 2003-12-04

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AU (1) AU2003240701A1 (fr)
DE (1) DE10222845A1 (fr)
WO (1) WO2003100047A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6080548A (en) * 1997-11-19 2000-06-27 Incyte Pharmaceuticals, Inc. Cyclic nucleotide phosphodiesterases
WO2000077040A2 (fr) * 1999-06-16 2000-12-21 Incyte Genomics, Inc. Molecules de signalisation intracellulaires
WO2001057190A2 (fr) * 2000-02-03 2001-08-09 Hyseq, Inc. Acides nucleiques et polypeptides
WO2001098471A2 (fr) * 2000-06-22 2001-12-27 Incyte Genomics, Inc. Phosphodiesterases

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6080548A (en) * 1997-11-19 2000-06-27 Incyte Pharmaceuticals, Inc. Cyclic nucleotide phosphodiesterases
WO2000077040A2 (fr) * 1999-06-16 2000-12-21 Incyte Genomics, Inc. Molecules de signalisation intracellulaires
WO2001057190A2 (fr) * 2000-02-03 2001-08-09 Hyseq, Inc. Acides nucleiques et polypeptides
WO2001098471A2 (fr) * 2000-06-22 2001-12-27 Incyte Genomics, Inc. Phosphodiesterases

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE EM_HUM 2 November 1998 (1998-11-02), HAYASHI M. ET AL.: "Homo sapiens cAMP-specific phosphodiesterase 8B (PDE8B) mRNA; partial cds" *
HAYASHI M ET AL: "Molecular cloning and characterization of human PDE8B, a novel thyroid-specific isozyme of 3',5'-cyclic nucleotide phosphodiesterase", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 250, no. 3, AF079529, 29 September 1998 (1998-09-29), pages 751 - 756, XP002091362, ISSN: 0006-291X *
HAYASHI MASAAKI ET AL: "Genomic organization, chromosomal localization, and alternative splicing of the human phosphodiesterase 8B gene.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. UNITED STATES 11 OCT 2002, vol. 297, no. 5, 11 October 2002 (2002-10-11), pages 1253 - 1258, XP002255295, ISSN: 0006-291X *
MARUYAMA ICHIRO N ET AL: "cRACE: A simple method for identification of the 5' end of mRNAs.", NUCLEIC ACIDS RESEARCH, vol. 23, no. 18, 1995, pages 3796 - 3797, XP008021587, ISSN: 0305-1048 *

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AU2003240701A1 (en) 2003-12-12

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