A process for producing exogenous protein in the milk of trangenic mammals and a process for purifying proteins therefrom

Abstract

The invention relates to a method of producing a protein of interest, comprising making a non-human transgenic mammal that produces said protein in its milk, obtaining said milk from the non-human transgenic mammal and purifying said protein of interest from the milk. Transgenic bovine animals were generated, which are able to produce human growth hormone in mammary glands. The method involves cloning of a genetic construct encoding hGH gene and beta casein promoter conveniently in an expression vector. It also includes transfection procedures into fetal bovine somatic cells, generally fibroblasts, and the nuclear transfer into enucleated bovine oocytes, generating thus transgenic embryos. The method also includes other procedures to generate transgenic embryos for the further expansion of the transgenic herd, such as the subcloning of transgenic female bovines, the superovulation of transgenic cows and their insemination with semen from a non-transgenic or a transgenic male bovine, and the superovulation of non-transgenic cows and their insemination with semen from a transgenic male bovine. Afterwards, transgenic embryos give rise to transgenic cattle that produce human growth hormone in huge amounts in their milk, from which the hormone is completely purified and analysed to fulfill all the requirements for the manufacture of a pure biopharmaceutical product.

Description

Background technique [0001] Protein factors and hormones involved in human health have been commonly produced by the pharmaceutical industry during the past decade either by extraction or by recombinant techniques. Genetic constructs involving the desired gene are successfully expressed in bacterial, yeast or mammalian cell lines. However, the use of mammalian cell cultures to obtain complex proteins, such as those requiring proper glycosylation patterns, involves costly methods. [0002] The past decade has seen increasing use of recombinant DNA technology to produce commercially important biological materials. To this end, DNA sequences encoding various medically important human proteins have been cloned. These include insulin, plasminogen activators, alpha 1 -antitrypsin and coagulation factors VIII and IX. Currently, even with emerging recombinant DNA techniques, these proteins are usually purified from blood and tissue, an expensive and time-consuming process that may ...

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Problems solved by technology

However, no polar body formation after NT entry into enucleated MII oocytes has been reported in cattle, sheep, or pigs, suggesting differences between species in the mechanically controlled formation of intact spindles and extrusion of polar bodies.

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