Ppl Therapeutics (scotland) Limited

Zymogenetics, Inc.

Pharming Intellectual Property Bv

Ppl Therapeutics

Ian Robert Cottingham

Ian Garner

Graham McCreath

Donald C Foster

Cindy A Sprecher

Donna E Prunkard

Simon M Temperley

A process for the production of a peptide is disclosed, the process comprising expressing in the milk of a transgenic, non-human, placental mammal a fusion protein which comprises the peptide to be expressed linked to a fusion partner protein which is lysozyme. The fusion protein may be separate from the milk and cleaved to yield the target peptide. A transgenic, non-human, placental mammal whose genome incorporates a DNA molecule comprising a coding sequence encoding lysozyme coupled to a peptide is also described.

Fusion proteins incorporating lysozyme

The present invention relates to the stabilization of milk from transgenic animals. In particular, the invention relates to the protection of proteins (e.g. fibrinogen) expressed in milk from transgenic animals by co-expression of a serine proteinase inhibitor (e.g., &#x3b1;-antitrypsin) in the milk of the transgenic animals.

Stabilization of milk from transgenic animals

Peptides can be produced in and purified from the milk of transgenic animals. The peptides are made as fusion proteins with a suitable fusion partner such as &agr;-lactalbumin, which is a natural milk protein. The fusion partner protein acts to promote secretion of the peptides and, at least in the case of &agr;-lactalbumin, allows a single-step purification based on specific affinity. The peptide is released from the purified fusion protein by a simple cleavage step and purified away from the now liberated &agr;-lactalbumin by repeating the same affinity purification method. A particular advantage of producing peptides via this route, in addition to the obvious advantages of high yield and biocompatibility, is that specific post-translational modifications, such as carboxy terminal amidation, can be performed in the mammary gland.

Peptide production as fusion protein in transgenic mammal milk

Methods for producing protein C in transgenic non-human mammals are disclosed. The protein C is modified at the two-chain cleavage site between the light and heavy chains of protein C from Lys-Arg to R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4 where R.sub.1 through R.sub.4 are individually Arg or Lys. DNA segments encoding modified protein C are introduced into the germ line of a non-human mammals, and the mammal or its female progeny produces milk containing protein C expressed from the introduced DNA segments. The protein C expressed from the introduced DNA segments has anticoagulant activity when activated. Non-human mammalian embryos and transgenic non-human mammals carrying DNA segments encoding heterologous protein C are also disclosed.

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Edinburgh, United Kingdom

Ian Robert Cottingham