Recombinant DNA method for production of parathyroid hormone

Abstract

A recombinant dna molecule coding for human parathyroid hormone operably linked to an inducible bacterial promoter, a leader growth hormone sequence, and a selective enzymatic cleavage site, expression of the molecule in bacteria, and methods for producing large quantities of human parathyroid hormone and its variants.

Description

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Human parathyroid hormone (hPTH) is a polypeptide of 84 amino acid residues (hPTH(1-84)). Its amino acid sequence (Keutmann, H. T. et al., Biochemistry 17:5723-5729 (1978)) and the nucleotide sequence of the gene coding for it (Handy et al. Proc. Natl Acad. Sci. USA 78;7365-7364 (1981)) are known.

The hPTH(1-84) nucleotide sequence can be obtained from a number of known sources. For example, the plasmid pPTHm124 (Born et al., Molec. Endocrinol. 1:5-14 (1987)) contains the sequence, and served as the source for the DNA used to construct the pGFP-1 plasmid of this invention (see FIG. 2).

The biological activities of hPTH, and methods for assaying these activities, are well known in the art (See, for example, Potts et al. Adv. Protein Chem. 32:323-395 (1982)). These activities include binding to receptors on kidney cells and on various tumor cell lines, and the stimulation of cyclic AMP production.

As used herein, a "variant" of hPTH is a polypeptide which possesses biological activity that is substantially similar to a biological activity hPTH. Such biological activity includes the binding to a receptor for hPTH and preventing the subsequent binding or action of hPTH at that receptor. A molecule is said to be "substantially similar" to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity. The "variants" of recombinant hPTH include both "fragments" and "variants" of recombinant hPTH.

The term "fragment of hPTH" is meant to refer to any polypeptide subset of that molecule. The term "variant of hPTH" is meant to refer to a molecule substantially similar in structure to either the entire molecule, or to a fragment thereof provided that the "variant" has at least one biological activity that is either similar to a biological activity of hPTH. Thus, provided that a molecule possesses at least one biological activity that is similar to an activity of hPTH (including the binding to a hPTH receptor and inhibiting the subsequent binding of hPTH), it is considered a "variant" of hPTH as that term is used herein, even if one of the molecules contains one or more amino acids not found in the other, or if the sequences of amino acid residues in the two molecules are not identical.

As contemplated in this invention, any variant of hPTH may be expressed and produced as long as it retains hPTH bioactivity, including the capacity to bind to hPTH receptors and function as a hPTH antagonist,

Variants of hPTH are produced by recombinant means. Truncated variants are produced by synthesizing polynucleotide expressible as polypeptides, that are missing amino acid residues from one or the other terminus.

The synthetic analog of bovine PTH, PTH (3-34) has been recognized as a potent PTH antagonist in vitro. Variants of hPTH lacking N-terminal amino acids 1-2 and 1-7, were shown to be devoid of agonist activity and capable of antagonist activity (Born, W. et. al., Endocrinol. 23:1848-1853 (1988)). Preferred variants of hPTH of this invention are variants truncated at the N-terminus. When a variant is truncated by one amino acid at the N -terminus, it is termed hPTH(2-84), in that it lacks residue #1 but contains residues #2-84. Likewise, variants truncated at the N-terminus are termed hPTH(3-84), hPTH(4-54), etc. The preferred variants of this invention are hPTH(384), (4-84), (5-84), (6-84), (7-84), and (8-84).

By the term "fusion protein" is intended a fused protein comprising human PTH, or a variant thereof, linked at its N-terminus to a "selective cleavage site," which is in turn, linked at its N-terminus to an additional amino acid leader polypeptide sequence. The linkage in the fusion protein is via conventional peptide bonds, produced in the host cell during protein biosynthesis, from the mRNA transcribed from the genetic sequences which comprises part of this invention.

The term "selective cleavage site" refers to an amino acid residue or residues which can be selectively cleaved with either chemicals or enzymes and where cleavage can be achieved in a predictable manner. A selective enzymatic cleavage site is an amino acid or a peptide sequence which is recognized and hydrolyzed by a proteolytic enzyme. Examples of such sites include trypsin or chymotrypsin cleavage sites. In a preferred embodiment of this invention, the selective cleavage site is comprised of the sequence Ile-Glu-Gly-Arg, which is recognized sad cleaved by blood coagulation factor Xa. In another embodiment, the selective cleavage site has the sequence Leu-Val-Pro-Arg, which is recognized and cleaved by thrombin.

The size of the oligonucleotide sequence 5' to the sequence coding for the enzyme recognition site can vary. In a preferred embodiment 13 nucleotides are situated between the codon for Ile (the start of the recognition site) and the 3' end of the leader sequence. In other embodiments longer sequences can be used. These upstream sequences must contain appropriate restriction sites to allow them to be cloned into the DNA adjacent to the leader sequence, as is the case for the BglII site of the oligonucleotide containing the Factor X recognition site (FXRS) (see FIGS. 1 and 2).

By the term "leader sequence" is intended a polynucleotide sequence linked to the hPTH, and expressed in host cells as n fusion protein fused to the selective cleavage site and the hPTH. The term "leader polypeptide" describes the expressed form of the "leader sequence" as obtained in the fusion protein.

In one embodiment, a leader sequence of no more than about 600 nucleotides is utilized. In another embodiment, a nucleotide sequence coding for human growth hormone (191 amino acid residues) is used, resulting in a fusion protein in which the leader polypeptide is human growth hormone (Ikehara et al., Proc. Natl. Acad. Sci. USA 81:5956-5960 (1984)). In another embodiment, the leader polypeptide is composed of about 50 amino acids. In yet another embodiment, the leader polypeptide is composed of about 100 amino acids.

The leader sequence as used in this invention can be less that the number of nucleotides required to code for a complete human growth hormone polypeptide, and can therefore code for a shortened variant of human growth hormone, as expressed in the fusion protein. In a preferred embodiment, the leader sequence comprises a nucleotide sequence coding for residues 1-138 of human growth hormone.

The fusion protein, which is often insoluble and found in inclusion bodies when it is overexpressed, is purified from other bacterial proteins by methods well known in the art. In a preferred embodiment, the insoluble fusion proton is centrifuged and washed after cell lysis, resolubilized with guanidine-HCl. It can remain soluble after removal of the denaturant by dialysis. (For purification of retractile proteins, sec Jones, U.S. Pat. No. 4,512,922; Olson, U.S. Pat. No. 4,518,526; Builder et al., U.S. Pat. Nos. 4,511,502 and 4,620,948.

The recombinant hPTH can be purified to be substantially free of natural contaminants from the solubilized fusion protein through the use of any of a variety of methodologies. As used herein, a compound is said to be "substantially free of natural contaminants" if it has been substantially purified from materials with which it is found following expression in bacterial or eukaryotic host cells. The hPTH may be purified through application of standard chromatographic separation technology.

Alternatively, the peptide may be purified using immuno-affinity chromatography (Rotman, A., et al., Biochim. Biophys. Acta 641:114-121 (1481); Sairam, M. R., J. Chromatog 215:143-152 (1981); Nielsen, L. S., et al., Biochemistry 21:6410-6415 (1982); Vockley, J., et al., Biochem. J. 217:535-542 (1984); Paucha, E., et al. J. Virol. 51:670-681 (1984); Chong, P., et al., J. Virol. Meth. 10:261-268 (1985)).

After partial or substantial purification, the fusion protein is treated enzymatically with the enzyme corresponding to the selective cleavage site. Alternatively, the fusion protein in its more impure state, even in refractile form, can be treated with the enzyme. If needed, the resulting mature hPTH, or variant thereof, can be further purified.

Conditions for enzymatic treatment are known to those of skill in the art.

The expression of a DNA sequence requires that the DNA sequence be "operably linked" to DNA sequences which contain transcriptional sad translational regulatory information. An operable linkage is a linkage in which the control or regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gent; expression. The precise nature of the "control regions" needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis. Regulatory regions in eukaryotic cells will in general include a promoter region sufficient to direct the initiation of RNA synthesis.

Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the fusion protein-encoding sequence or (3) interfere with the ability of the fusion protein-encoding sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of transcribing that DNA sequences.

To express the hPTH molecule (or a variant or functional derivative thereof) in a prokaryotic cell (such as, for example, E. coli. B. subtilis. Pseudomonas. Streotomyces, etc.), it is necessary to operably link the hPTH-encoding sequence to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the β-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pPR325, etc. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (P_L and P_R), the trp, recA, lacZ, lacI. and gal promoters of E. coli. the α-amylase (Ulmanen, I., et al., J. Bacterial. 162:176-182 (1985)) and the σ-28-specific promoters of B. subtilis (Gilman, M. Z., et al., Gene 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T. J., In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streotomyces promoters (Ward, J. M., et al., Mol. Gen. Genet. 203:468-478 (1986)). Prokaryotic promoters are reviewed by Glick, B. R., (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)).

The joining of various DNA fragments, to produce the expression vectors of this invention is performed in accordance with conventional techniques, employing blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkali and phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. The genetic construct encodes an inducible promoter which is operably linked to the 5' gene sequence of the fusion protein, to allow efficient expression of the fusion protein.

The preferred prokaryotic promoter for this invention is the E. coli trp promoter, which is inducible with indole acrylic acid.

Proper expression in a prokaryotic cell requires the presence of a ribosome binding site upstream of the gene-encoding sequence. Such ribosome binding sites are disclosed, for example, by Gold, L., et al. (Ann. Rev Microbiol. 35:365-404 (1981)).

If expression is desired in a eukaryotic cell, such as yeast, fungi, mammalian cells, or plant cells, then it is necessary to employ a promoter capable of directing transcription in such a eukaryotic host. Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Homer, D., et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C., et al., Nature (London) 290:304-310 (1981)); and the yeast ga14 gene promoter (Johnston, S. A., et al., Proc. Natl Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P. A., et al., Proc. Natl. Acad. Sci. (USA) 81:5931-5955 (1984)).

Preferably, the introduced pane sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as; for example, pBR322, ColEl, pSC101, pACYC 184, π VX. Such plasmids are, for example, disclosed by Maniatis, T., et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982)). Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pIJIOI (Kendall, K. J., et al., J. Bacterial. 169:4177-4183 (1987)), and streptomycin bacteriophages such as φC31 (Chester, K. F., et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, J. F., et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki, K. (Jon. J. Bacterial. 33:729-742 (1978)).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc. Such plasmids are well known in the art (Holstein, D., et al., Miami Wntr. Symp. 19:265-274 (1982); Broach, J. R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, J. R., Cell 28:203-204 (1982); Bollon, D. P., et al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise. Vol. 3, Gene Expression, Academic Press, N.Y., pp. 563-608 (1980)).

The preferred bacterial host for this invention is E. coli. In other embodiments, other bacterial species can be used. In yet other embodiments, eukaryotic cells may be utilized, such as, for example, yeast, filamentous fungi, or the like. Use of these cell types are well known in the art. Any host tray be used to express the protein which is compatible with replicon and control sequences in the expression plasmid. In general, vectors containing replicon and control sequences are derived from species compatible with a host cell are used is connection with the host. The vector ordinarily carries a replicon site, as well as specific genes which are capable of providing phenotypic selection in infected or in transformed cells. The expression of the fusion protein can also be placed under control with other regulatory sequences which may be homologous to the organism in its untransformed state.

hPTH can then be used for all of its well known uses.

EXAMPLE 1

PRODUCTION OF HUMAN PTH(1-84)

E. coli transformed with pGFP-1 (see FIG. 2) in 10 ml were inoculated into a liter of LB ampicilline medium. When the culture reached an optical density of 600 nm, 40 mg. of 3β-indoleacrylic acid were added and the cultures induced for 16 hr. at 37°C C. Cells were pelleted by centrifugation at 5000×g for 10 min, were resuspended in 100 ml of TE-sucrose to which 100 mg of lysozyme was added. After 1.5 hrs. incubation at 0°C C., MgCl₂ was added to a concentration of 20 mM and DNaseI to 50 μg/ml for a further 1.5 hrs. The cells were lysed by addition of 200 ml of a solution containing 0.2M NaCl, 1% deoxycholic acid, 1.6% Nonidet P40, 20 mM Tris-HCl (pH 7.3), 2 mM EDTA, and 0.5 mM dithiothreitol (DTT), and incubation for 1.5 hrs at 0°C C. The lysate was pelleted at 5000×g for 10 min. The pellet was washed 4 times with 0.5% Triton X-100, mM EDTA, and 0.5 mM DTT, and a pellet enriched in fusion protein was obtained by centrifugation.

The fusion protein pellet was dissolved in 50 ml 6M guanidine-HCL, 20 mM Tris-HCl, (pH 7.5), 0.5 mM DTT, and 1 mM EDTA, such that the fusion protein was at 1 mg/ml. The solution was dialyzed against 50 mM Tris-HCl (pH 7.8), 0.1M NaCl.

Activated Factor X was added at a ratio of 1 mg per 100 mg fusion protein and the mixture incubated for 2 hrs. at 37°C C. The solution was then applied to an HPLC RPC-18 system, and was eluted with a linear 30-70% acetonitrile/H2O gradient. The PTH fraction was found to elute at 38% acetonitrile. The fraction was lyophilized and resuspended in water for subsequent analysis.

It was found that the GH-PTH fusion protein was hydrophobic and eluted at 68%a acetonitrile. Following treatment with Factor Xa, the hybrid protein peak was markedly diminished with the concomitant appearance of the PTH peak at 38% acetonitrile.

Yields of hybrid GH-PTH were generally about 50 mg/liter of culture. By SDS-polyacrylamide gel electraphoretic analysis, the above conditions of Factor X treatment resulted in 50% conversion of the fusion protein to hPTH(1-84) and the GH fragment.

EXAMPLE 2

ANALYSIS OF AMINO ACID COMPOSITION AND N-TERMINAL SEQUENCE OF RECOMBINANT HPTH(1-84)

Amino acid compositional analysis corresponded to the expected amino acid composition for hPTH(1-84). Gas phase microsequencing of the hPTH(1-84) confirmed the sequence for the first 18 amino acids. Significantly, N-terminal position 1 was found to be serine. There was no evidence of blocked amino termini in a significant portion of the hPTH produced, as has been described by others (Rabbani et al., 1988, supra).

EXAMPLE 3

BIOLOGICAL ACTIVITIES OF RECOMBINANT HPTH(1-84)

The recombinant peptide produced as described in Example 1 above was tested for it binding to two cells types. In FIG. 3 is shown data indicating that recombinant hPTH(1-84) bound specifically to opossum kidney cells, with half-maximal binding occurring at about 3 nM. This was similar to the binding of synthetic PTH(1-34) which demonstrated half-maximal binding at about 1 nM FIG. 4 depicts similar studies using the UMR rat osteosarcoma cell line, wherein the recombinant hPTH(1-84) bound with a half-maximum value of about 10 nM, compared to about 2 nM for synthetic PTH(1-34).

The biological activity of inducing cyclic AMP production is shown in FIGS. 5 and 6. Recombinant hPTH(1-84) stimulated cyclic AMP in opossum kidney cells with an EC₅₀ of about 10 nM, and synthetic PTH(1-34) had an EC₅₀ of about 4 nM (FIG. 5). In UMR cells recombinant hPTH(1-84) stimulated cyclic AMP with an EC₅₀ of about 3 nM, whereas synthetic PTH(1-34) had an EC₅₀ of about 0.1 nM (FIG. 6). Therefore, the recombinant peptide had PTH biological activity which is largely indistinguishable from that of a known active fragment.

DEPOSIT

E. coli strain DHl containing the plasmid pGFP-1 was deposited at the American Type Culture Collection, Rockville, MD, on May 12, 1989 and was given the accession number ATCC 67977.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

Claims

1. A recombinant dna molecule comprising:

(a) an expression control region, said region in operable linkage with:

(b) a leader sequence coding for human growth hormone amino acids 1-138 operably linked to:

(c) a nucleotide sequence coding for a selective enzymatic cleavage site, said nucleotide sequence being linked to and preceding a genetic sequence coding for human parathyroid hormone (hpth) or a polypeptide variant thereof having the capacity to bind to a receptor for hpth.

2. The molecule of claim 1 wherein said control region includes a bacterial, viral, fungal, or mammalian promoter.

3. The molecule of claim 2 wherein said promoter is selected from the group consisting of the right promoter of bacteriophage λ (P_L), the left promoter of bacteriophage λ (P_R), the E. coli trp promoter, the E. coli recA promoter, the E. coli lacZ promoter, the E. coli lacI promoter, the E. coli gal promoter, the B. subtilis α-amylase promoter, the B. subtilis α-28-specific promoter, the promoters of the bacteriophages of Bacillus, and the Streptomyces promoters.

4. The molecule of claim 3 wherein said promoter is the E. coli trp promoter.

5. The molecule of claim 1 wherein said selective enzymatic cleavage site is cleaved by Factor Xa.

6. The molecule of claim 5 wherein said selective enzymatic cleavage site is Ile-Glu-Gly-Arg.

7. The molecule of claim 1 wherein said selective enzymatic cleavage site is cleaved by thrombin.

8. The molecule of claim 1 wherein said selective enzymatic cleavage site is Leu-Val-Pro-Arg.

9. The molecule of claim 8 wherein said human parathyroid hormone is hpth (1-84).

10. The molecule of claim 8 wherein said polypeptide variant thereof having the capacity to bind to a receptor for hpth is truncated at the N-terminus.

11. The molecule of claim 10 wherein said polypeptide variant thereof having the capacity to bind to a receptor for hpth is selected from the group consisting of hpth(2-84), hpth(3-84), hpth(4-84), hpth(5-84), hpth(6-84), hpth(7-84), and hpth(8-84).

12. The recombinant dna molecule of claim 1 which is a plasmid.

13. The plasmid of claim 12 which is pGFP-1.

14. A host cell containing the molecule of claim 1.

15. The cell of claim 14 which is prokaryotic.

16. The cell of claim 15 which is bacterial.

17. The cell of claim 16 which is of the species E. coli.

18. The cell of claim 14 which is eukaryotic.

19. The cell of claim 18 which is a yeast cell or a mammalian cell.

20. A method of producing human parathyroid hormone (hpth) or a polypeptide variant thereof having the capacity to bind to a receptor for hpth, substantially free of natural contaminants, comprising:

(a) providing the molecule of claim 7 in a host;

(b) expressing said human parathyroid hormone or a polypeptide variant thereof having the capacity to bind to a receptor for hpth thereof; and

(c) obtaining a fusion protein containing a human growth hormone, fused to a selective enzymatic cleavage site, fused to hpth(1-84) or a polypeptide variant thereof having the capacity to bind to a receptor for hpth; and

(d) cleaving said fusion protein of step (c) with an enzyme recognizing said selective enzymatic cleavage site.

21. The method of claim 20 further comprising:

(f) purifying said hpth(1-84) or said polypeptide variant thereof having the capacity to bind to a receptor for hpth.

22. A fusion protein comprising:

(a) a human growth hormone 1-138 fused to

(b) a selective enzymatic cleavage site, fused to

(c) hpth(1-84) or a polypeptide variant thereof having the capacity to bind to a receptor for hpth.

23. The protein of claim 22 wherein said selective enzymatic cleavage site is cleaved by Factor Xa.

24. The protein of claim 22 wherein said selective enzymatic cleavage site is Ile-Glu-Gly-Arg.

25. The protein of claim 22 wherein said selective enzymatic cleavage site is cleaved by thrombin.

26. The protein of claim 22 wherein said selective enzymatic cleavage site is Leu-Val-Pro-Arg.

27. The protein of claim 22 wherein said polypeptide variant thereof having the capacity to bind to a receptor for hpth is selected from the group consisting of hpth(2-84), hpth(3-84), hpth(4-84), hpth(5-84, hpth(6-84), hpth(7-84), and hpth(8-84).

28. The protein of claim 27 wherein said polypeptide variant thereof having the capacity to bind to a receptor for hpth is hpth(3-84).

29. The method of claim 21 wherein said hpth(1-84) is purified using an HPLC RPC-18 system and is eluted with a linear 30-70% acetonitrile/H₂O gradient.

30. human parathyroid hormone substantially free of natural contaminants produced according to the method of claim 29.