Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • 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
  • C07K14/57527—Calcitonin gene related peptide
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
  • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
  • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

• the present invention relates generally to the manipulation of genetic materials and, more particularly, to the manufacture of specific DNA sequences useful in recombinant procedures to secure the production of human calcitonin and polypeptide analogs thereof.
• Calcitonin is a 32 amino acid polypeptide hormone secreted by parafollicular ("C") cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish.
• C parafollicular
• calcitonin is the product of in vivo cleavage from a higher molecular weight precursor molecule.
• Calcitonins from eight species have been purified to homogeneity and the Structures of seven forms have been determined. Among these seven structurally-characterized calcitonins is human calcitonin, which has an amino acid sequence as follows: 5 10
• calcitonin In man, a major role of calcitonin is to protect the skeleton during periods of calcium stress, pregnancy and lactation. Direct renal effects and actions on the gastrointestinal tract have also been . noted. Salmon calcitonin appears to have biological actions essentially identical to calcitonins of mammalian origin, but its potency per mg is greater and it has a longer duration of action. Consequently, synthetic salmon calcitonin, rather than human calcitonin, is currently employed therapeutically in the treatment of Paget's disease and hypercalcemia.
• the base sequence includes one or more codons selected from among alternative codons specifying the same amino acid on the basis of preferential expression characteristics of the codon in a projected host microorganism, e.g., E. coli.
• Other preferred forms of manufactured genes include those wherein: (1) base codons specify additional amino acids in the polypeptide synthesized which facilitate isolation of biologically active human calcitonin (e.g., an initial Met residue, or Lys-Arg sequence); and/or (2) base codons specifying human.
• calcitonin are preceded and/or followed by a sequence of bases comprising a portion of a base sequence which provides for restriction endonuclease cleavage of a DNA sequence (e.g., a PstI site) and consequently facilitates formation of expression vectors.
• a sequence of bases comprising a portion of a base sequence which provides for restriction endonuclease cleavage of a DNA sequence (e.g., a PstI site) and consequently facilitates formation of expression vectors.
• a manufactured gene capable of directing the synthesis in a selected host microorganism of a human calcitonin polypeptide analogs which differ from human calcitonin polypeptide in terms of the identity and/or location of one or more amino acids (e.g., [Val 8 ] human calcitonin and [Asn 15 ] human calcitonin); and
• a fusion gene comprising a manufactured gene according to the invention fused to a second gene capable of directing synthesis of a second polypeptide (e.g.,
• DNA sequences including manufactured genes are inserted into a viral or circular plasmid DNA vector to form a hybrid vector and the hybrid vectors are employed to transform host micro-organisms such as bacteria (e.g., E. coli) or yeast cells.
• the transformed microorganisms are thereafter grown under appropriate nutrient conditions and express the polypeptide products of the invention.
• Novel DNA sequences of the invention are preferably synthesized from nucleotide bases according to the methods disclosed in the aforementioned co- owned, concurrently-filed U.S. Patent Application Serial No. 375,493, by Yitzhak Stabinsky, entitled “Manufacture and Expression of Structural Genes" (Attorney's Docket No. 6250). Briefly summarized, the general method comprises the steps of:
• each duplex strand including a double stranded region of 12 or more selected complementary base pairs and further including a top single stranded terminal sequence of from 3 to 7 selected bases at one end of the strand and/or a bottom single stranded terminal sequence of from 3 to 7 selected bases at the other end of the strand, each single stranded terminal sequence of each duplex DNA strand comprising the entire base complement of at most one single stranded terminal sequence of any other duplex DNA strand prepared;
• each duplex DNA strand prepared in step (1) to one or two different duplex strands prepared in step (1) having a complementary single stranded terminal sequence, thereby to form a single continuous double stranded DNA sequence which has a duplex region of at least 27 selected base pairs including at least three base pairs formed by complementary association of single stranded terminal sequences of duplex DNA strands prepared in step (1) and which has from 0 to 2 single stranded top or bottom terminal regions of from 3 to 7 bases.
• At least three different duplex DNA strands are prepared in step (1) and all strands so prepared are annealed concurrently in a single annealing reaction mixture to form a single continuous double stranded DNA sequence which has a duplex region of at least 42 selected base pairs including at least two non-adjacent sets of 3 or more base pairs formed by complementary association of single stranded terminal sequences of duplex strands prepared in step (1).
• the duplex DNA strand preparation step (1) of the DNA sequence manufacturing process noted above preferably comprises the steps of: (a) constructing first and second linear deoxyoligonucleotide segments having 15 or more bases in a selected linear sequence, the linear sequence of bases of the second segment comprising the total complement of the sequence of bases of the first segment except that at least one end of the second segment shall either include an additional linear sequence of from 3 to 7 selected bases beyond those fully complementing the first segment, or shall lack a linear sequence of from 3 to 7 bases complementary to a terminal sequence of the first segment, provided, however, that the second segment shall not have an additional sequence of bases or be lacking a sequence of bases at both of its ends; and,
• sequence of bases in the double stranded DNA subunit sequences formed preferably includes one or more triplet codons selected from among alternative codons specifying the same amino acid on the basis of preferential expression characteristics of the codon in a projected host microorganism, such as yeast cells or bacteria, especially E. coli bacteria.
• the term "manufactured" as applied to a DNA sequence or gene shall designate a product either totally chemically synthesized by assembly of nucleotide bases or derived from the biological replication of a product thus chemically synthesized.
• the term is exclusive of products "synthesized” by cDNA methods or genomic cloning method ologies which involve starting materials which are initially of biological origin.
• nucleotide bases A for adenine; G for guanine; T for thymine; U for uracil; and C for cytosine.
• Table I provides a tabular correlation between the 64 alternate triplet nucleotide base codons of DNA and the 20 amino acids and transcription termination ("stop") function specified thereby.
• deoxyoligonucleotides Preparation of deoxyoligonucleotides is carried out according to the general methodologies published in Mutteucci, et al., J. Am. Chem. Soc., 103, pp. 3185-3192 (1981) and Beaucage, et al., Tetrahedron Letters, 22, pp. 1859-1862 (1981) and the references cited therein.
• the synthesis begins by derivatizing high performance liquid chromatography grade silica gel to contain appropriately protected nucleotides.
• the deoxyoligonucleotides are linked through the 3'- hydroxyl group to a carboxylic acid functional group attached covalently to the silica gel.
• the chemical steps used for the addition of one nucleotide to this support are as follows: (1) detritylation using ZnBr 2 in nitromethane/methanol (4 min.); (2) condensation of a 5'-di-p-anisylphenyl- methyl deoxynucleoside 3'-methoxy-N, N-dimethylamino- phosphine with the support bound nucleoside (5 min.); (3) blocking unreacted support bound nucleoside hydroxyl groups with acetic anhydride (5 min.); and (4) oxidation of the phosphite to the phosphate with I 2 (2 min.). Syntheses are performed in simple sintered glass funnels by a single technician.
• the time required for one synthetic cycle is 20 to 30 minutes and deoxyoligonucleotides containing up to 30 mononucleotides may be obtained in high yields in less than 15 hours.
• the redundancy of the genetic code is capitalized upon to avoid the formation, in any given deoxyoligonucleotide, of widely separated base sequences which are the complement of each other, thereby enhancing yields of desired linear strands by avoiding opportunities for the strands to "fold over" on themselves through base complementation.
• the projected host for expression of the DNA sequences manufactured was an E. coli microorganism, wherever possible, alternative codon selection was based on E. coli codon preferences.
• each polymer (100 mg, containing 2-5 micromoles of oligonucleotide) is treated with 2 ml of thiophenol: Et 3 N:dioxane (1:2:2) solution for 75 min. at room temperature. After washing, this step is followed by treatment with concentrated ammonium hydroxide at 20°C for 2 hours to hydrolyze the ester joining the deoxyoligonucleotides to the support. After centrifugation and recovery of the supernatant containing the deoxyoligonucleotides, the base protection groups are removed by warming in a sealed tube at 50°C for 24 hours. The ammonium hydroxide solution is then evaporated to dryness.
• the deoxyoligonucleotides are desalted on a Sephadex G50/40 column (45 x 2.5 cm) using 10 mM TEAB (ph 7.0). Recoveries from 10 O.D. units of crude DNA generally range from 1.0 to 2.0 O.D. units (260 nm).
• the following example illustrates the preparation of a DNA sequence which comprises a gene coding for [Lys -2 , Arg -1 ] human calcitonin and which includes terminal base sequences factilitative of insertion of the sequence into the ⁇ -lactamase gene of pBR322 at the Pst I site about 540 base pairs downstream of the ⁇ -lactamase initiation codon.
• the annealed pairs were mixed according to the following scheme: DE1,DE7 and 1,5; 2,6 and 3,7; and 4,8 and DE3,DE4. Mixtures were warmed to 37° for 10 minutes and cooled to 4° for 2 hours. To the three cool mixtures were added T4-DNA ligase (New England Biolabs, 3 Weiss units), DTT and ATP to give a final concentration of 20mM DTT and .8mM AT. The ligation was allowed to proceed at 4° for 12 hours. The reaction mixtures were boiled to denature the ligase. Table II illustrates the double stranded
• the reaction mixutres were boiled to denature the ligase.
• the reaction mixture after cooling was sent through a 10ml column of Sephadex G-150-40.
• the fractions containing the desired ligation products were pooled, dried, resuspended in 20 microliters ligation buffer and treated with ligase as before.
• the final ligation product contained a band corresponding to 112 bases on urea-polyacrylamide gel.
• the band was excised and DNA was electroeluted, ethanol precipitated, resuspended with buffer and ligated into M13 mp8 or mp9 duplex DNA.
• Bacterial cells were infected and single stranded DNA was sequenced, verifying the base sequence illustrated in Table II.
• the manufactured DNA sequence was ligated into an expression vector (pBR322) and the hybrid vector was employed to transform E. coli cells. Such cells were grown in culture under appropriate nutrient conditions. Expression of the desired biologically active product may be verified by radioimmunoassay of cell products using a commerically available R1A kit (e.g., "Calcitonin II I 125 R1A Kit", Catalog No. 2500, Immuno Nuclear corp., Stillwater, Minnesota).
• R1A kit e.g., "Calcitonin II I 125 R1A Kit", Catalog No. 2500, Immuno Nuclear corp., Stillwater, Minnesota.
• Deoxyoligonucleotides were prepared as in Example 2 except that segments DE 4 and DE 5 were omitted and variant segements DE 5 and DE 6 were prepared and employed in their place in the segment anneal ing reaction.
• the alternate segment base sequences were:
• Segments DE 1 and DE 6 were not kinased. Pairs of DNA strands were allowed to anneal by boiling, followed by gradual cooling: DE1,DE7; 1,5; 2,6; 3,7; 4,8; and DE5,DE6. The annealed pairs were mixed according to the following scheme: DE1,DE7, and 1,5; 2,6 and 3,7; and 4,8 and DE5,DE6. Mixtures were warmed to 37° for 10 minutes and cooled to 4° for 2 hours. To the three cool mixtures were added T4-DNA ligase (New England Biolabs, 3 Weiss units), DTT and ATP to give a final concentration of 20mM DTT and .8mM ATP. The ligation was allowed to proceed at 4° for 12 hours. The reaction mixtures were boiled to denature the ligase. The reaction mixture after cooling was sent through a 10 ml column of Sephadex G-150-40.
• the fractions containing the desired ligation products were pooled, dried, resuspended in 20 microliters ligation buffer and treated with ligase as before.
• the final ligation product contained a band corresponding to 121 bases on urea-polyacrylamide gel. The band was excised and DNA was eluted, precipitated, resuspended and ligated into a verification vector as in Example 2.
• the base sequence of the DNA sequence manufactured was verified and the sequence was inserted in pBR322. Expression of the desired product by E. coli cells transformed with the hybrid vector may be verified by radioimmunoassay.
• the following example illustrates the prepara tion of a manufactured DNA sequence which comprises a gene coding for [Ala -2 , Met -1 , Val 8 ] human calcitonin which includes "blunt ended" terminal portions comprising base pair recognition sites for Pst I restriction endonuclease cleavage.
• terminal portions may be subjected to Pst I treatment prior to projected insertion into an expression vector at a Pst I site.
• Deoxyoligonucleotides were prepared as in Example 2, except that segments 1, 2 and DE 1 through DE 4 were omitted and replaced by segments CA1, CA3, and CA4 through CA7 in the annealing reaction.
• the oligonucleotides prepared and annealed were as follows
• CA 1 5'-TGCGGTAACCTGTCTACCTGCGT-3'
• CA 3 5'-CAGCACGCAGGTAGACAGGTTAC-3'
• CA 4 5'-CGGCTGCAGCAATG
• CA 5 5'-CGCACATTGCTGCAGCCG
• CA 6 5'-TGATAACTGCAGCCG
• Pairs of DNA strands were allowed to anneal by boiling, followed by gradual cooling: CA4,CA5; CA1,CA3; 2,6; 3,7; 4,8; and CA6,CA7.
• the annealed pairs were mixed according to the following scheme: CA4,CA5 and CA1,CA3; 2,6 and 3,7; and 4,8 and CA6,CA7.
• Mixtures were wamred to 37° for 10 minutes.
• the three mixtures were then combined to give one mixture which was warmed at 37° for an additional 10 minutes then cooled to 4° for 2 hours.
• T4-DNA ligase New England Biolabs, 3 Weiss units
• DTT and ATP to give final concentration of 20mM DTT and .8mM ATP.
• the ligation was allowed to proceed at 4° for 12 hours.
• the reaction mixtures were boiled to denature the ligase.
• the reaction mixture after cooling was. sent through a 10ml column of Sephadex G-150-40.
• the final ligation product contained a band corresponding to 125 bases on urea-polyacrylamide gel.
• the band was excised and DNA was eluted, precipitated and resuspended as in Example 2. It has not as yet been cloned in a verification vector.
• the DNA sequence putatively assembled is set forth in Table III below.
• any alternative amino acid codon providing the desired initial G-C pair could have been employed (e.g., GTT, specifying valine).
• GTT specifying valine
• the following example illustrates the preparation of a DNA sequence which comprises a gene coding for [Met -1 , Val 8 ] human calcitonin which includes an initial base sequence providing a six base recogni tion site for cleavage by Xba I restriction.
• endonuclease and bases providing a portion of a six base EcoRI restriction endonuclease recognition site, together with a terminal sequence providing a six base recognition site for cleavage by Bg1 II and a portion of a six base BamHI restriction site.
• CA 1 5'-TGCGGTAACCTGTCTACCTGCGT-3' CA 3 5'-CAGCACGCAGGTAGACAGGTTAC-3' CA 15 5'-AATTCTCTAGAATG-3' CA 16 5'-CGCACATTCTAGAG-3' CA 17 5'-TGATAGATCTG-3' CA 18 5'-GATCCAGATCTATCACGGA-3'
• Pairs of DNA strands were allowed to anneal by boiling, followed by gradual cooling: CA15,CA16;
• the annealed pairs were mixed, warmed to
• the following example illustrates the preparation of a DNA sequence which comprises a gene coding for [Ala -2 , Glu -1 , Asn 15 ] human calcitonin which includes terminal base sequences providing a portion of the six base recognition site for retriction endonuclease EcoRI at one end of the DNA as well as a portion of the six base recognition site for Pstl restriction endonuclease at the other end of the DNA.
• AAC asparagine-specifying codon
• polypeptide products include those of Examples 2 and 3 having the Lys-Arg sequence preceding cystine at amino acid position number 1 which are initially synthesized in the microorganisms as constituents of a fusion protein.
• suitable protease will yield polypeptides having the "native" [Cys 1 ] initial residue.
• Suitable proteases include the enzyme commercially available as "Submaxillaris Protease” (Pierce Chemical Co., Rockford, Illinois).
• Trypsin may also be employed, provided suitable steps are taken to avoid protease action at the lysine present at amino acid position number 18.
• Other examples of a polypeptide product is that of Example 3 having a Gly-Lys-Lys-Arg sequence following proline at amino acid sequence number 32. Products of this type will, upon treatment with carboxamide group-forming proteases endogenous in calcitonin synthesizing organisms, provide products having the terminal Pro-C-NH 2 group present in forms of human calcitonin which have higher biological activity.
• C-terminal carboxamide is not provided in polypeptides of the invention, it may be formed by methanolic ammonia treatment which can be expected to form a carboxamide group at other acidic residues present (e.g., change an aspartic acid present at amino acid position 15 to asparagine).]
• Polypeptide analogs of human calcitonin having glutamine preceding [Cys 1 ] as illustrated in Example 6 may be treated with Staph A V-8 protease to restore cystine as the initial amino acid.
• polypeptide products of the invention include those having an initial methionine preceding the "native" [Cys 1 ], as in Examples 4 and 5. Treatment of such products with cyanogen bromide will restore cystine as the initial amino acid in the polypeptide.
• the present invention provides human calcitonin analogs wherein an "internal" amino acid of the native sequence is varied.
• Such compounds include the [Val8.] analogs of Examples 4 and 5 and the [Asn 15 ] compound of Example 6.
• Products of the present invention and/or antibodies thereto may be suitably "tagged", for example radiolabelled (e.g., with I 125 ) conjugated with enzymes or fluorescently labelled, to provide reagent materials useful in assays and/or diagnostic test kits, for the qualitative and/or quantitative determination of the presence of such products and/or said antibodies in fluid samples.
• Such antibodies may be obtained from the innoculation of one or more animal species (e.g., mice rabbit, goat, human, etc.) or from monoclonal antibody sources. Any of such reagent materials may be used alone or in combination with a suitable substrate, e.g., coated on a glass or plastic particle or bead.
• Novel DNA sequences of the invention illustrated in the above examples typically include all or part of a base sequence providing a recognition site for DNA cleavage by restriction endonuclease enzymes which is facilitative of insertion into expression.

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• 1983
  • 1983-04-15 EP EP19830901772 patent/EP0107710A4/fr not_active Withdrawn
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