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
1987-09-15
2001-07-03
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S254200, C435S320100, C435S325000, C435S349000, C530S350000, C536S023500
Reexamination Certificate
active
06255067
ABSTRACT:
TECHNICAL AREA OF THE INVENTION
The field of this invention is post-translational processing enzymes, more particularly the enzyme known as peptidyl-glycine alpha-amidating monooxygenase (PAM).
BACKGROUND OF THE INVENTION
Small, biologically active peptides are frequently produced as larger, usually inactive precursors (preprohormones). Specific proteolytic cleavages liberate various peptides from these precursors. A variety of covalent modifications of the precursor molecule and the smaller peptides occur following synthesis of the preprohormone. One such modification is alpha-amidation, in which the carboxy-terminal carboxylic acid group of a peptide becomes blocked with an alpha-amide group. Approximately half of all known bioactive peptides contain amide groups at their C-termini. In most cases, unblocked versions of these peptides are much less active (on the order of 0.1 to 1%) than their amidated derivatives.
While it is possible to synthesize by chemical means small peptides which contain an amide group at the C-terminal end (alpha-amide), larger alpha-amidated peptides are difficult and expensive to produce. Larger peptides are often produced by expression in bacteria or yeast, but these microbes have not been shown to contain enzymes which can catalyze peptide alpha-amidation of expressed peptides. Thus many peptides produced in cultured microbial cells require the action of PAM to achieve full activity.
PAM activity has been detected in porcine, bovine, human and rat pituitary as well as in other species and tissues, such as frog skin. Some of these PAM enzymes have been purified or partially purified. See, for example, Murthy, et al., Journal of Biological Chemistry, Vol. 261, pp. 1815-1822 (1986) and Mizuno, et al., Biochem. Biophys. Res. Comm., Vol. 137, pp. 984-991 (1986). Prior art purification methods of PAM activity have been directed to soluble enzymes from tissue extracts. This has led to the identification of proteins of approximate molecular weight of 60,000 in porcine pituitaries and molecular weights of 38,000 and 54,000 from bovine pituitaries. However, studies have shown that there is very little PAM protein isolatable from natural sources such as bovine and porcine pituitaries. Thus there is a need in the art to obtain a ready source of PAM enzyme.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a DNA isolate which encodes a PAM enzyme.
It is an object of the present invention to provide a DNA vector which encodes a PAM enzyme.
It is another object of the present invention to provide PAM proteins containing membrane spanning domains.
It is yet another object of the present invention to provide a method for producing a PAM enzyme in cultured cells.
It is still another object of the present invention to provide a method of activating peptide hormone precursors to their mature amidated forms using a PAM preparation.
These and other objects are provided by one or more of the following embodiments of the present invention. In one embodiment a DNA vector is provided which encodes a PAM enzyme. In another embodiment a DNA isolate is provided which encodes a PAM enzyme.
In another embodiment, a method of producing a PAM enzyme is presented which comprises providing cultured cells which can replicate and express an intron-free coding sequence of a PAM enzyme, growing said cultured cells, and recovering the PAM produced from said cultured cells. The transformed cells themselves are also contemplated by the present invention.
In yet another embodiment of the present invention PAM proteins comprising a membrane spanning domain are provided which are substantially free of other proteins which do not have PAM activity.
In yet another embodiment of the present invention an in vitro method is provided of activating a hormone precursor to produce a mature hormone having an alpha-amide group on the C-terminal amino acid residue, said method comprising, providing a peptide hormone precursor having a glycine residue on the C-terminal side of the C-terminal amino acid residue of the mature hormone, and contacting said peptide hormone precursor with a membrane preparation having PAM activity to form an alpha-amidated derivative of the peptide hormone.
The present invention provides the art with a ready source of PAM protein similar to that purified from natural sources (e.g., pituitary) as well as its precursor forms and a membrane associated form. By producing PAM protein in cultured cells, one can obtain much larger amounts of protein than are currently practicably available from natural sources. In addition, the existence of a membrane associated form of the PAM enzyme allows for the use of immobilized PAM enzyme in the in vitro maturation and processing of genetically engineered hormones and bioactive peptides.
REFERENCES:
patent: 4708934 (1987-11-01), Gilligan
patent: 0299790 (1989-01-01), None
Bradbury et al, Nature, vol. 298, 1982, pp. 686-688.
Eipper et al, Proceedings of the National Academy of Sciences USA, vol. 80, (1983), pp. 5144-5148.
Bradbury et al, Biochemical and Biophysical Research Communications, vol. 112, (1983), pp. 372-377.
Bradbury et al, “C-Terminal Amide Formation In Peptide Hormones”, Biogenetics of Neurohormonal Peptides, 1985, pp. 171-186.
Murthy et al, The Journal of Biological Chemistry, vol. 261, 1986, pp. 1815-1822.
Murthy et al, Molecular Endocrinology, vol. 1, 1987, pp. 290-299.
Mehta et al., “Purification of a Peptidylglycine alpha-Amidating Enzyme from Transplantable Rat Medullary Thyroid Carcinomas,” Arch. Biochem & Biophys. (1988), 261:44-54.
Perkins et al., “Stable Expression of the Catalytic Domain of Bovine Pituitary Peptidylglycine Alpha-Amidating Monooxygenase (PAM) cDNA in AtT-20 Mouse Pituitary Corticotropes,” Endocrinology (1989), 124:262a, Abstract 958.
May et al., “Membrane-Associated Forms of Peptidylglycine alpha-Amidating Monooxygenase Activity in Rat Pituitary,” J. Biol. Chem. (1988), 263:7550-7554.
Eipper Betty
Keutmann Henry T.
Mains Richard
Rodriguez Henry
Schofield Peter
Banner & Witcoff Ltd
Schwartzman Robert A.
The Johns Hopkins University
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