Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-08-19
2002-01-08
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069400, C435S320100, C435S254200, C530S399000, C530S350000, C536S023500
Reexamination Certificate
active
06337194
ABSTRACT:
The present invention concerns double-chain disulfide-bonded molecules, particularly insulin, and precursor molecules for same, together with DNA sequences coding for same, processes for preparation of said precursors, and processes for the preparation of the molecule.
Human insulin is a non-steroidal hormone comprising two polypeptide chains (A and B); the A-chain comprising 21 amino acid residues (A
1−2
) and the B-chain comprising 30 amino acid residues (B
1−30
) The A- and B-chains are joined by two intermolecular disulfide bridges. A third intramolecular disulfide bridge is formed within the A-chain.
Human insulin is naturally produced in the pancreas by the &bgr;-cells of the islets of Langerhans, via. a single 110 amino acid precursor polypeptide (preproinsulin) (Chan, S. J. et al., 1976, Proc. Natl. Acad. Sci. USA, 73: 1964-1968; Sheilds and Blobel, 1977, Proc. Natl. Acad. Sci. USA, 74: 2059-2063) with a structure of:
(NH
2
) pre-peptide-B-chain-C-peptide-A-chain (COOH)
The human preproinsulin (precursor) undergoes various post-translational modifications and events to convert it into mature insulin. The first step is removal of the prepeptide (Bell, G. I. et al., 1979, Nature 282: 525-527), which acts as a signal sequence to direct the molecule (proinsulin) upon synthesis into the endoplasmic reticulum (ER) and hence into the secretory pathway. After entry into the ER, the resultant proinsulin then folds and the three disulfide bridges are formed (Chan et al., 1976, supra; Lomedico, P. T. et al., 1977, J. Biol. Chem., 259: 7971-7978; Shields and Bloebel, 1977 supra). The proinsulin then passes to the Golgi, is packaged into secretory granules and is converted into mature insulin by endoproteolytic cleavage (Steiner, D. F. et al., 1984, J. Cell. Biol., 24: 121-130; Tager and Steiner, 1974, Ann. Rev. Biochem., 43: 509-538).
Since the discovery of insulin in 1921, the nature of insulin preparations used to treat diabetics has shown a steady evolution (Owens, D. R., 1986, Human Insulin, pp 5-33 MTP Press). A human source of insulin has always been impractical due to low yields from the pancreas and degradation. However, the structure of insulin is highly conserved in other mammals, making it possible to use other animals as a source of insulin. This has led to the development of porcine and bovine insulins. However, they are difficult to manufacture, great care having to be taken to ensure purity and to minimise their allergic response.
Latterly, recombinant DNA methods have allowed the synthesis of various forms of recombinant human insulin. This has been achieved using
E. coli
and
Saccharomyces cerevisiae
. Early techniques involved the production of separate A- and B-chains (Goeddel, D., et al., 1979, Proc. Natl. Acad. Sci. USA, 76: 106-110; Chance, R. E. et al., 1981, In: Rich, D. H. & Gross, E. (eds.) Peptides: Synthesis-Structure-Function, Proc. Seventh American Peptide Symposium, pp 721-728, Rockford II, Pierce Chemical Co.; Frank, B. H. et al., 1981, In. Rich, D. H. & Gross, E. (eds.) Peptides: Synthesis-Structure-Function, Proc. Seventh American Peptide Symposium, pp 729-738. Rockford II, Pierce Chemical Co.; Steiner, D. F., et al., 1968, Proc. Natl. Acad. Sci. USA, 60: 622; and EP-A-0 090 433).
However, these procedures, all of which require the chemical combination of the A- and B-chains, have several serious drawbacks. One is that the fusion proteins accumulate intracellularly and are subject to proteolytic degradation. They must all be purified from the other intracellular materials, and
E. coli
materials are pyrogenic. Additionally, chemical dusulfide bond formation is inefficient.
An alternative approach has been to produce insulin from eukaryotic cells and utilise the secretion pathway to modify precursor insulin into the mature form as happens in the pancreatic &bgr;-cells and also to secrete the product into the culture medium, away from the intracellular proteins, where there are few contaminants from which it needs to be purified. Examples of such work include EP 0 121 884 A; EP 0 195 691 A; Wollmer, A., et al., 1974, Hoppe-Seyler's Z. Physiol. Chem., 355: 1471-1476; Brandenburg, D. et al., 1973, Hoppe-Seyler's Z. Physiol. Chem., 354:1521-1524; Thim, L., et al., 1986, Proc. Natl. Acad. Sci. USA, 83: 6766-6770; Thim, L., et al., 1987, FEBS, Let 212: 307-312; EP 0 163 529 A; EP 0 427 296 A; Markussen, J., et al., 1986, (In Peptides, 1986, Theodoropoulos, D., (ed.) pp. 189-194, Proc. 19th Eur. Peptide Symp. on Peptide, Porto Carras-Chalkidiki, Greece. Walter de Gruyter & Co, New York) and EP 0 347 845 A.
FIGS. 1 and 2
show a mini-proinsulin (Thim, L. et al., 1986, supra).
However, these are unable to give high yields of mature insulin or near-mature insulin, instead being primarily concerned with producing high levels of insulin precursors (for example, insulin precursors with the carboxy-terminus residue of the B-chain (B
30
) missing) which subsequently require costly and extensive chemical alteration in order to convert them into mature insulin. The present invention overcomes the limitations and disadvantages of the prior art and provides simple, convenient and economic double-chain molecules and precursor molecules, in particular insulin, together with DNA sequences coding for same, processes for preparation of said precursors, and processes for the preparation of insulin and insulin analogues.
According to the present invention there is provided a protein precursor for at least two polypeptide chains having the general formula B-Z-A wherein B and A are the two polypeptide chains of a double-chain molecule, the two chains being linked by at least one disulfide bond, and Z is a polypeptide comprising at least one proteolytic cleavage site.
The precursor may be produced in a host. By ‘host’ is meant a system which is capable of producing the protein precursor of the present invention. The host may be cells of a single- or multi-cellular organism, or it may be a cell-free system. For example, the host may be eukaryotic. It may be yeast or fungal cells or it may be an animal, for example sheep, rat or mouse, or it may be a cell-line from an animal.
Alternatively, the precursor may be produced in a cell-free host system.
Proteolytic cleavage of Z may produce the double-chain molecule, possibly in its mature form, or a near precursos thereof.
The double-chain molecule may be insulin, the B and A polypeptides representing, respectively, the B- and A-chains of insulin.
Insulin may for example be human, bovine or porcine insulin or a partially modified form thereof For example, modification may be by way of addition, deletion or substitution of amino acid residues. Substitutions may be conserved substitutions. Modification of human insulin to produce porcine insulin may be achieved by substitution of alanine at residue B
30
. Bovine insulin may be produced from human insulin by substitution of alanine at residue B
30
, of alanine at A
8
and of valine at A
10
. Partially modified forms of molecules (comprising amino acid residues or nucleic acids) may be considered to be homologues of the molecules from which they were derived. The may have at least 50% homology with the molecules from which they were derived. They may for example have at least 60, 70, 80, 90 or 95% homology.
The present inventors have found that, surprisingly, despite the problems associated with the prior art synthesis of recombinant insulin, mature insulin and near-precursors of insulin may be produced in vivo in organisms such as yeast using genetic constructs, the mature insulin resulting from post-translational processing of the precursor molecule. Moreover, these insulin molecules may be produced at high yield by yeast, making the present invention an economically viable alternative to the present methods of synthesising insulin.
The polypeptide Z may also comprise at least one additional polypeptide. Hence not only may a double-chain molecule such as insulin be produced, but an additional molecule or molecules, which may also require post-translational pr
Hadfield Christopher
Krishnaswamy Patnam R
Meacock Peter A
Raina Krisha K
Ramadoss Candadai S
Jones Day Reavis & Pogue
O'Hara Eileen B.
Spector Lorraine
Vittal Mallya Scientific Research Foundation
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