Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in Aspergillus

• US 6,080,559 A
• Filed: 06/29/1998
• Issued: 06/27/2000
• Est. Priority Date: 05/05/1989
• Status: Expired due to Term

• Current Assignee: Agennix, Inc. (Agennix AG)
• Original Assignee: Agennix, Inc. (Agennix AG)
• Inventors: Headon, Denis R., O'Malley, Bert W., Conneely, Orla M.
• Primary Examiner(s): Achutamurthy, Ponnathapu
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US09/107,075
• Time in Patent Office: 729 Days
• Field of Search: 530/350, 530/300, 530/400, 530/412, 435/69.7, 435/254.21, 435/254.23, 435/254.3, 435/320.1, 435/252.3, 536/23.2, 536/23.5
• US Class Current: 435/69.7
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/79 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/80 for fungi

Abstract

The subject invention provides for the production of lactoferrins and lactoferrin polypeptide fragments using the host cells Aspergillus in combination with novel plasmid constructs. More specifically, the subject invention provides novel vector constructs capable of producing lactoferrins and lactoferrin polypeptide fragments in Aspergillus host cells. More particularly, the subject invention provides for novel plasmid constructs suitable for use with Aspergillus and especially Aspergillus awamori, niger and oryzae host cells, which enables them to produce large amounts of recombinant lactoferrins and lactoferrin polypeptide fragments.

Claims

1. An intact, deglycosylated lactoferrin protein or a single domain, deglycosylated lactoferrin polypeptide fragment produced by a process that comprises culturing a transformed Aspergillus fungal cell containing a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':

(a) a promoter;

(b) a nucleotide sequence encoding a signal peptide;

(c) a 5'"'"' portion of a nucleotide sequence of a gene encoding an amino-terminal portion of a highly expressed endogenous, secreted Aspergillus polypeptide;

(d) a nucleotide sequence encoding a peptide linker, said peptide linker comprising a cleavage site of a protease endogenous to Aspergillus; and

(e) a nucleotide sequence encoding lactoferrin or lactoferrin polypeptide fragment;

wherein said transformed Aspergillus fungal cell is cultured in a suitable nutrient medium until a lactoferrin protein or a lactoferrin polypeptide fragment is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein said processed lactoferrin or lactoferrin polypeptide fragment is secreted into the nutrient medium and isolated therefrom and wherein the lactoferrin protein or the lactoferrin polypeptide fragment has been deglycosylated.

2. An intact, deglycosylated lactoferrin protein or a single domain, deglycosylated lactoferrin polypeptide fragment according to claim 1 wherein the lactoferrin protein or lactoferrin polypeptide fragment is selected from the group consisting of human lactoferrin, porcine lactoferrin and bovine lactoferrin.

3. An intact, deglycosylated lactoferrin protein or a single domain, deglycosylated lactoferrin polypeptide fragment according to claim 1 wherein the lactoferrin protein or lactoferrin polypeptide fragment is human lactoferrin.