WO1983004028A1

WO1983004028A1 - The manufacture and expression of genes for calcitonin and polypeptide analogs thereof

Info

Publication number: WO1983004028A1
Application number: PCT/US1983/000562
Authority: WO
Inventors: Edward Pak-Tung Lau; Sidney V. Suggs
Original assignee: Applied Molecular Genetics, Inc.
Priority date: 1982-05-06
Filing date: 1983-04-15
Publication date: 1983-11-24
Also published as: IT8367473A0; EP0107710A1; IL68490A0; EP0107710A4; IT1212981B

Abstract

DNA sequences comprising structural genes coding for (1) a polypeptide having the amino acid sequence and properties of human calcitonin and for (2) polypeptide analogs thereof which differ in terms of the identity and/or location of one or more amino acids, e.g., ADVal8 BD and ADAsn15 BD analogs of human calcitonin. Structural gene sequences may be provided with initial and terminal sequences which facilitate production of discrete protein products by selected host microorganisms as well as for expression by host organisms of fusion proteins, e.g., beta-lactamase-calcitonin and beta-galactosidase-calcitonin from which the desired products may be isolated. DNA sequences may also be provided with codons specifying additional initial and/or terminal amino acid sequences in polypeptides expressed, which additional amino acids facilitate isolation of active proteins.

Description

THE MANUFACTURE AND EXPRESSION OF GENES FOR CALCITONIN AND POLYPEPTIDE ANALOGS THEREOF

BACKGROUND

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.

Incorporated by reference herein for the purpose of providing information pertinent to the prior art with respect to recombinant DNA techniques is 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).

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. As is the case with other polypeptides displaying hormonal activity (e.g., ACTH and endorphin) 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

H₂N-Cys-Gly-Asn-Leu-Ser-Thr -Cys-Met-Leu-Gly-Thr-Tyr-Thr-

15 20 25

Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-

30 Ile-Gly-Val-Gly-Ala-Pro-CONH₂

[See, generally, "Calcitonin 1980, Chemistry, Physiology, Pharmacology and Clinical Aspects", A. Pecile, Ed., Exerpta Medica, Amsterdam (1981)]

In all biologically active forms of calcitonin isolated to date, there is a disulfide "bridge" linking the cystine residues in positions 1 and 7 and a prolinamide group at the polypeptide's carboxyl terminus. On the basis of tests of structurally modified polypeptides, both of these structural features appear to be essential for significant levels of biological activity.

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.

Unfortunately, some patients treated with salmon calcitonin develop severe systemic hypersensitivity reactions to the heterologous species hormone, and local inflammatory reactions at the administration site have been reported for fully ten percent of patients, Some patients with Paget's disease who have good biochemical and symptomatic responses to salmon calcitonin initially, later relapse, possibly due to formation of neutralizing antibodies. Circulating antibodies to salmon calcitonin after 2 to 18 month's treatment have been reported in about half the Paget's disease patients in whom antibody tests have been performed. Occasionally, patients with extremely high antibody titers are found and these usually will have suffered a relapse and will be unresponsive to the hypocalcemic effects of salmon calcitonin.

Attempts have been made to develop analogs of human calcitonin which will possess the high potency and duration of effects of salmon calcitonin but which will not display the adverse immunological effects noted above. The systematic synthesis and testing of polypeptide analogs has been hindered to a great extent by the absence of efficient method for their production. Small quantities of analogs can be synthesized by the well-known Merrifield procedure [Merrifield, J.Am.Chem.Soc, 85, pp. 2149-2154 (1963)], but the quantities produced and the time needed for preparation of given analogs make such procedures unsatisfactory. The construction and partial characterization of recombinant plasmids containing human calcitonin cDNA sequences has recently been reported [Craig, et al., Nature, 295. pp. 345-347 (1982)]. Recombinant DNA techniques for the manufacture, cloning and expression of a structural gene for human calcitonin and genes for polypeptide analogs which differ therefrom in terms of the identity and/or location of one or more amino acids have not been brought to bear on this problem.

BRIEF SUMMARY

Provided by the present invention is a manufactured gene capable of directing synthesis in a selected host microorganism of human calcitonin. In a preferred form of manufactured gene, 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.

Also provided by the present invention are:

(1) 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., [Val⁸] human calcitonin and [Asn¹⁵] human calcitonin); and

(2) 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.,

(3-lactamase and β-galactosidase) in a manner permitting the synthesis of a fused polypeptide including human calcitonin polypeptide or a human calcitonin polypeptide analog. In practice of the invention to generate polypeptide products, 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:

(1) preparing two or more different, linear, duplex DNA strands, 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; and

(2) annealing 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.

In the preferred general process of manufacture, 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,

(b) combining the first and second segments under conditions conducive to complementary association between segments to form a linear, duplex DNA strand.

The 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.

Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description thereof. DETAILED DESCRIPTION

As employed herein, 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. As such, 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.

The following abbreviations shall be employed herein to designate amino acids: Alanine, Ala; Arginine, Arg; Asparagine, Asn; Aspartic acid, Asp; Cystein, Cys; Glutamine, Gin; Glutamic acid, Glu; Glycine,

Gly; Histidine, His; Isoleucine, lie; Leucine, Leu; Lysine, Lys; Methionine, Met; Phenylalanine, Phe; Proline, Pro; Serine, Ser; Threonine, Thr; Tryptophan, Trp; Tyrosine, Tyr; Valine, Val. The following abbreviations shall be employed for nucleotide bases: A for adenine; G for guanine; T for thymine; U for uracil; and C for cytosine.

For ease of understanding of the present invention. Table I below 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.

The following example illustrates a preferred general procedure for preparation of deoxyoligonucleo tides for use in the manufacture of DNA sequences of the invention.

EXAMPLE 1

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 ZnBr₂ 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 I₂ (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. Whenever possible, 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. Further, because 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. [See, e.g., Grantham, et al., Nucleic Acids Research, 8, pp. r49-r62 (1980); Grantham, et al., Nucleic Acids Research, 8, pp. 1893-1912 (1980); and, Grantham, et al., Nucleic Acids Research, 9 , pp. r43-r74 (1981)]. Purification and isolation of the deoxyoligonucleotide is completed using the following procedure. After the final condensation step including removal of the terminal di-p-anisyl-phenylmethyl group with acid, the silica gels containing the deoxyoligonucleotides are washed thoroughly with methanol and air dried. To remove the methyl group from the phosphotriesters, each polymer (100 mg, containing 2-5 micromoles of oligonucleotide) is treated with 2 ml of thiophenol: Et₃N: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 residue is redissolved in 2 ml of H₂O, filtered and the solution then washed three times with n-butanol (3 x 2 ml). Final purification of the product is by electrophoresis on 20% polyacrylamide gels using a tris-borate buffer (pH 8) containing 7 M urea. Approximately 10 O.D. units

(260 nm) of the crude material in 30 μls of 80% formamide is loaded into a well 2.5 cm wide and electrophoresis is conducted at 16 v/cm. To detect DNA bands, the gel is placed on a fluorescent TLC plate (Sigma Chemical Company) and illuminated with long UV light in order to visualize the deoxyoligonucleotides which appear as intensely absorbing bands in each lane. The desired band is cut from the gel and the DNA eluted with 0.5 M ammonium acetate, 10 mM MgCl₂, 0.1% SDS, and 0.1mM EDTA. After two washings with n-butanol, 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 β-lactamase gene of pBR322 at the Pst I site about 540 base pairs downstream of the β-lactamase initiation codon.

EXAMPLE 2

The following deoxyoligonucleotides were synthesized according to the procedures of Example

1. starting with silica gel-linked 5'-dimethoxytrityl deoxynucleotide:

1. 5'-TGCGGTAACCTGTCTACCTGCAT-3'

2. 5' -GCTGGGCACCTATACTCAGGACTT-3 '

3. 5'-CAACAAATTCCATACCTTCCCGC-3'

4. 5'-AGACCGCTATCGGTGTTGGTGCTCCG-3'

5. 5'-CAGCATGCAGGTAGACAGGTTAC-3' 6. 5'-GTTGAAGTCCTGAGTATAGGTGCC-3'

7. 5'-GGTCTGCCGGGAAGGTATGGAATTT-3'

8. 5'-GCACCAACACCGATAGC-3' DE 1 5'-AAACGC-3'

DE 3 5'-TGATGATGCA-3' DE 4 5'-TCATCACGGA-3'

DE 7 5'-CGCAGCGTTTTGCA-3'

0.5 nmol aliquots of the completely deprotected DNA in dry form were dissolved in 20 microliter of kination buffer. Phosphorylation of the 5'-OH end was catalyzed by T4 polynucleotide kinase using 32P-ATP and cold carrier ATP. DE1 and DE4 were not kinased to avoid unwanted ligation product later on.

The extent of kinations were monitored by ion-exchange paper chromatography on Whatman DE-81 strips. After the kinations had gone to completion, the reaction mixtures were boiled to denature the kinase. 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 DE3,DE4. 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

DNA sequence obtained as a product of the deoxyoligonucleotide annealing reaction recited above. Individual segments are designated by brackets and amino acids specified by the codons formed are shown above the sequence. It may be noted that, on the sequence shown, Pst I "sticky ends" (3'-A C G T-5' and 5'-T G C A-3') were formed at the terminal regions of the sequence, but the sequence did not include remaining bases for the six base Pst I recognition site. Thr Cys M A C C T G C A ' T G G A C G T

20 His Thr P C A T A C C T G T A T G G A

Stop

DE3 AT G AT G C A TA C T-5 ^•

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.

After verification of the base sequence, 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 I¹²⁵ R1A Kit", Catalog No. 2500, Immuno Nuclear corp., Stillwater, Minnesota).

The following example illustrates preparation of a DNA sequence which comprises a gene coding for

[Lys^-2, Arg^-1, Gly³³, Lys³⁴, Lys³⁵, Arg³⁶] human calcitonin which includes terminal base sequences facilitative of insertion of the sequence into the β-lactamase gene of pBR322 as previously noted.

EXAMPLE 3

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:

DE 5 5'-GGTAAGAAGCGCTGATGCA-3' DE 6 5'-TCAGCGCTTCTTACCCGGA-3'.

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 sequence of the DNA, as verified, was idential to that prepared in Example 2 except that the region coding for the terminal amino acids was as follows (starting with codons for alanine at amino acid position 31):

31 32 33 34 35 36 Ala Pro Gly Lys Lys Arg Stop

The following example illustrates the prepara tion of a manufactured DNA sequence which comprises a gene coding for [Ala^-2, Met^-1, Val⁸] human calcitonin which includes "blunt ended" terminal portions comprising base pair recognition sites for Pst I restriction endonuclease cleavage. For ease of hybrid vector preparation, such terminal portions may be subjected to Pst I treatment prior to projected insertion into an expression vector at a Pst I site.

EXAMPLE 4

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

2. 5' -GCTGGGCACCTATACTCAGGACTT-3 '

3. 5'-CAACAAATTCCATACCTTCCCGC-3'

4. 5'-AGACCGCTATCGGTGTTGGTGCTCCG-3' 6. 5'-GTTGAAGTCCTGAGTATAGGTGCC-3'

7. 5'-GGTCCTGCGGGAAGGTATGGAATTT-3'

8. 5'-GCACCAACACCGATAGC-3'

CA 1 5'-TGCGGTAACCTGTCTACCTGCGT-3' CA 3 5'-CAGCACGCAGGTAGACAGGTTAC-3' CA 4 5'-CGGCTGCAGCAATG

CA 5 5'-CGCACATTGCTGCAGCCG CA 6 5'-TGATAACTGCAGCCG

CA 7 5'-CGGCTGCAGTTATCACGGA

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. To the cool mixture were added 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. It may be noted that, because the projected function of the initial Alaspecifying codon was to provide a G-C base pair useful in developing a reconstitutable six base recognition site for Pst I cleavage, any alternative amino acid codon providing the desired initial G-C pair could have been employed (e.g., GTT, specifying valine).

The following example illustrates the preparation of a DNA sequence which comprises a gene coding for [Met^-1, Val⁸] 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.

EXAMPLE 5

The following deoxyoligonucleotides were prepared as in Example 2:

2. 5' -GCTGGGCACCTATACTCAGGACTT-3 '

3. 5'-CAACAAATTCCATACCTTCCCGC-3'

4. 5'-AGACCGCTATCGGTGTTGGTGCTCCG-3' 6. 5'-GTTGAAGTCCTGAGTATAGGTGCC-3'

7. 5'-GGTCCTGCGGGAAGGTATGGAATTT-3'

8. 5'-GCACCAACACCGATAGC-3'

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;

CA1,CA3; 2,6; 3,7; 4,8; and CA17,CA18.

The annealed pairs were mixed, warmed to

37° for 10 minutes and cooled to 4° for 2 hours. To the cooled mixture 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 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 kinase as before. The final 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 cloned sequence has not as yet been verified. The putative sequence is set forth in Table IV below.

The following example illustrates the preparation of a DNA sequence which comprises a gene coding for [Ala^-2, Glu^-1, Asn¹⁵] 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.

EXAMPLE 6

Following the procedures generally described in the previous examples, a DNA sequence was prepared, cloned and verified which included an asparagine-specifying codon (AAC) at a site coding for the amino acid in position number 15 and which differs from the sequence shown in Table II in the manner illustrated below.

It will be apparent from the above illustrative examples that a variety of novel DNA sequences and polypeptide products are provided according to the invention. Examples of 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. In such a case. treatment of the fusion protein with a suitable protease will yield polypeptides having the "native" [Cys¹] 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-NH₂ group present in forms of human calcitonin which have higher biological activity. [Where such a 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 [Cys¹] as illustrated in Example 6 may be treated with Staph A V-8 protease to restore cystine as the initial amino acid.

Still other polypeptide products of the invention include those having an initial methionine preceding the "native" [Cys¹], as in Examples 4 and 5. Treatment of such products with cyanogen bromide will restore cystine as the initial amino acid in the polypeptide.

Along with the above-noted polypeptide analogs of human calcitonin wherein one or more amino acids precedes or follows the native sequence, 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 [Asn¹⁵] 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.

Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing illustrative examples. Consequently, the invention should be considered as limited only to the extent reflected by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A manufactured gene capable of directing the synthesis in a selected host microorganism of human calcitonin polypeptide.

2. A manufactured gene according to claim 1 wherein 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.

3. A manufactured gene according to claim 1 wherein 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 E. coli .

4. A manufactured gene according to claim

1 wherein the base sequence comprises the following:

5'-T G C G G T A A C C T G T C T A C C T G C A T G C T G- 3'-A C G C C A T T G G A C A G A T G G A C G T A C G A C-

G G C A C C T A T A C T C A G G A C T T C A A C A A A T- C C G T G G A T A T G A G T C C T G A A G T T G T T T A-

T C C A T A C C T T C C C G C A G A C C G C T A T C G G T- A G G T A T G G A A G G G C G T C T G G C G A T A G C C A-

G T T G G T G C T C C G-3' C A A C C A C G A G G C-5'

5. A manufactured gene according to claim 1 wherein base codons specifying human calcitonin include initial and/or terminal codons respectively specifying additional initial and/or terminal amino acids in the polypeptide synthesized which facilitate isolation of biologically active human calcitonin.

6. A manufactured gene according to claim

1 wherein the base codons specifying human calcitonin are preceded and/or followed by a sequence of bases comprising a portion of a base sequence which provides a recognition site for restriction endonuclease enzyme cleavage.

7. A manufactured gene capable of directing the synthesis in a selected host microorganism of a human calcitonin polypeptide analog which differs from human calcitonin polypeptide in terms of the identity and/or location of one or more amino acids.

8. A manufactured gene according to claim

7 wherein 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.

9. A manufactured gene according to claim

8 wherein 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 E. coli.

10. A manufactured gene according to claim 7 wherein the base sequence comprises one of the following: 5'-T G C G G T A A C C T G T C T A C C T G C G T G C T G- 3'-A C G C C A T T G G A C A G A T G G A C G C A C G A C-

T C C A T A C C T T C C C G C A G A C C G C T A T C G G- A G G T A T G G A A G G G C G T C T G G C G A T A G C C-

T G T T G G T G C T C C G-3' A C A A C C A C G A G G C-5'

and

G G C A C C T A T A C T C A G A A C T T C A A C A A A T- C C G T G G A T A T G A G T C T T G A A G T T G T T T A-

T G T T G G T G C T C C G-3' A C A A C C A C G A G G C-5'

11. A manufactured gene according to claim 7 wherein base codons specifying human calcitonin include initial and/or terminal codons respectively specifying additional initial and/or terminal amino acids in the polypeptide synthesized which facilitate isolation of a biologically active human calcitonin analog.

12. A manufactured gene according to claim 7 wherein the base codons specifying human calcitonin are preceded and/or followed by a sequence of bases comprising a portion of a base sequence which provides a recognition site for restriction endonuclease cleavage of a DNA sequence.

13. A fusion gene comprising a manufactured gene according to either of claims 1 to 7 fused to a second gene capable of directing synthesis of a second polypeptide in a manner permitting the synthesis of a fused polypeptide inlcuding human calcitonin polypeptide or a human calcitonin polypeptide analog.

14. A fusion gene according to claim 13 wherein said second gene is a gene directing synthesis of β-galactosidase enzyme.

15. A fusion gene according to claim 13 wherein said second gene is a gene directing synthesis of β-lactamase.

16. A biologically functional DNA microorganism transformation vector including a manufactured gene according to claim 1.

17. A biologically functional DNA microorganism transformation vector including a manufactured gene according to claim 7.

18. A biologically functional DNA microorganism transformation vector including a fusion gene according to claim 13.

19. A vector according to either of claims 16, 17 or 18 which is a circular DNA plasmid.

20. A microorganism transformed with a vector according to either of claims 16, 17 or 18.

21. A proces for the production of human calcitonin polypeptide comprising: growing, under appropriate nutrient conditions, microorganisms transformed with a biologically functional DNA including a manufactured gene according to claim 1, whereby said microorganisms express said gene and produce human calcitonin polypeptide.

22. A process according to claim 21 wherein the microorganisms grown are E. coli microorganisms.

23. A process for the production of human calcitonin polypeptide analog comprising: growing, under appropriate nutrient conditions, microorganisms transformed with a biologically functional DNA including a manufactured gene according to claim 7, whereby said microorganisms express said gene and produce human calcitonin polypeptide.

24. A process according to claim 21 wherein the microorganisms grown are E. coli microorganisms.

25. A polypeptide product of the expression in a microorganism of a manufactured gene according to claim 1.

26. A polypeptide product of the expression in a microorganism of a manufactured gene according to claim 7.

27. A product according to claim 26 which is [Val⁸] human calcitonin.

28. A product according to claim 26 which is [Asn ¹⁵] human calcitonin.

29. A reagent material comprising a radio-labelled manufactured gene according to claim 1.

30. A reagent material according to claim

29 wherein said radiolabel is I ¹²⁵

31. A reagent material comprising a tagged antibody to a polypeptide according to claim 25, coated on the surface of a plastic bead.