Peptide production as fusion protein in transgenic mammal milk

Details Peptide production as fusion protein in transgenic mammal milk Peptide production as fusion protein in transgenic mammal milk

1997-09-25

2001-03-06

Clark, Deborah J.R. (Department: 1633)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S023500, C800S004000, C800S007000, C435S455000

Reexamination Certificate

active

06197946

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the production of peptides in the milk of transgenic mammals, particularly non-human placental mammals.
2. Related Art
Polymers of naturally occurring amino acids concatenated via their amino and carboxyl groups form the basis of many different biologically important compounds. Polymers of 3 to 100 amino acids are generally called peptides whilst larger concatamers are termed proteins. This is a purely arbitrary distinction, and the term “peptide” will be generally used throughout this specification even though the definition of peptides is not restricted to polymers of any particular size. Peptides can be biologically active without further modification or they can form the building blocks for more complex molecules by chemical incorporation into larger structures or by modification such as glycosylation. The term “peptide” is used herein to include biologically active or inactive molecules which may or may not be further modified by either chemical methods or in biological systems.
The direct chemical synthesis of peptides is expensive due to the cost of reagents and the high degree of purification needed to remove failed sequences. Microbial synthesis by recombinant DNA technology is not always appropriate for peptides, because of difficulties in their extraction and purification and the absence in the microbial host of enzymes for performing appropriate and correct post-translational modification. Heterologous proteins can be produced in stably transfected mammalian cell lines. Many such cell lines are available today and are used commercially, but concern remains that the cell lines were in general established from tumours of various types. More recently, the production of proteins in the milk of transgenic mammals such as sheep has become a reality, as illustrated in WO-A-8800239 and WO-A-9005188.
This invention relates to an economical process for the bulk production of peptides in the milk of transgenic animals. The production of peptides in milk is ideal as a bulk process because very large volumes of milk can be harvested using simple and environmentally safe technology. A second advantage of using transgenic technology is that only biologically safe materials are produced. This is in contrast to chemical methods where side reactions may produce toxic materials which can only be removed at additional cost.
Another advantage of using a biological process is that some reactions which can be essential for biological activity, for example carboxy-terminal amidation, are difficult to perform in good yield by chemical means. Carboxy-terminal amidation is catalysed by a specific enzyme which recognises and modifies peptides or proteins with a glycine residue at the carboxy terminus (“Peptidylglycine &agr;-Amidating Monooxygenase: A Multifunctional Protein with Catalytic, Processing and Routing Domains” Eipper, B. A et al. (1993) Protein Science 2, 489-497). Therefore, suitably designed proteins will be specifically amidated before secretion into the milk of producer animals. This is only one example of a range of post-translational modifications which can be carried out by the biosynthetic pathways in the mammary gland and which can potentially be harnessed for the synthesis of particular peptide entities. Other examples of desirable post-translational modifications include disulphide bridge formation, &ggr;-carboxylation of glutamic acid residues and the addition of O- and N-linked glycosylation (“In Vivo Chemical Modification of Proteins”, Wold, F.,
Ann. Rev. Biochem.
50 783-814 (1981)).
The technology for producing large quantities of recombinant proteins, as opposed to shorter peptides, in milk is well established. The human protease inhibitor &agr;
1
-antitrypsin, for example, has been produced in the milk of transgenic sheep at levels in excess of thirty grams of protein per liter (“High Level Expression of Active Human &agr;
1
-Antitrypsin in the Milk of Transgenic Sheep” Wright, G. et al. (1991)
Bio/Technology,
9 77-84). It is expected that the same technology can be applied to the production of proteins in cattle which can routinely produce up to 10,000 liters of milk per lactation.
BRIEF SUMMARY OF THE INVENTION
Production of proteins in the milk of transgenic producer animals is extremely advantageous in that, providing the protein is actually secreted by the mammary gland into the milk, no cellular extraction step is necessary. Nonetheless, the protein in question does, in many applications of the technology, have to be extracted from the milk produced, and it is to this problem that the present invention is particularly addressed. The invention also addresses the problem of the production of peptides, particularly relatively short peptides, whose properties may be such that they would normally interfere with their production.
DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of the invention, there is provided a process for the production of a peptide, the process comprising expressing in the milk of a transgenic non-human placental mammal a fusion protein comprising the peptide linked to a fusion partner protein, separating the fusion protein from the milk and cleaving the fusion protein to yield the peptide.
The reasons for producing the desired peptide as a fusion protein are essentially three-fold. First, it is expected that the established technology for producing relatively large proteins in milk will be applicable to the production of corresponding fusion proteins in which a peptide has been fused to the original protein. A second function of the fusion partner is to disguise properties of the peptide which might otherwise interfere with its production. Thirdly, the fusion partner may facilitate purification from milk by providing the peptide as part of a larger molecule. Milk is a complex biological fluid which contains fats, sugars, proteins and also peptides and proteolytic fragments, so the purification of synthetic peptides from such a mixture would be complex and expensive.
The use of living organisms as the production process means that all of the material produced will be chemically identical to the natural product. In terms of basic amino acid structures this means that only L-optical isomers, having the natural configuration, will be present in the product. Also the number of wrong sequences will be negligible because of the high fidelity of biological synthesis compared to chemical routes, in which the relative inefficiency of coupling reactions will always produce failed sequences. The absence of side reactions is also an important consideration with further modification reactions such as carboxy-terminal amidation. Again, the enzymes operating in vivo give a high degree of fidelity and stereospecificity which cannot be matched by chemical methods. Finally the production of peptides in a biological fluid means that low-level contaminants remaining in the final product are likely to be far less toxic than those originating from a chemical reactor.
Peptides producible by the invention are preferably from 3 to 100 amino acid residues in length, but the invention is not limited to the production of peptides of the preferred size range. The invention is particularly appropriate for producing &agr;-amidated or other post-translationally modified peptides. Many peptides found in the nervous and endocrine systems of animals and bioactive peptides from other sources which have actions on the nervous system are &agr;-amidated. Examples include the following, which are:
&agr;-amidated residue
A alanine
b,o CRH; p Galanin; &mgr;-Conotoxin
C cysteine
crustacean cardioactive peptide; conotoxins G1, M1,
S1
D aspartic
deltorphin
E glutamic
joining peptide
F phenylalanine
FMRF-NH
2
; gastrin; cholecystokinin; CGRP; &ggr;
1
MSH
G glycine
oxytocin; vasopressin; GnRH; pancreastatin;
leucokinin I, II; Manduca adipokinetic hormone;
leucokinin I, II
H histidine
Apamin; scorpion toxin II
I isoleucine
h,r CRH; PHI; Manduca diuretic hormone; rat
neuropeptide EI (melani

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