WO1991005045A1 - SEQUENCE D'ADNc ET D'ACIDE AMINE DE LACTOFERINE PORCINE - Google Patents

SEQUENCE D'ADNc ET D'ACIDE AMINE DE LACTOFERINE PORCINE Download PDF

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Publication number
WO1991005045A1
WO1991005045A1 PCT/US1990/005245 US9005245W WO9105045A1 WO 1991005045 A1 WO1991005045 A1 WO 1991005045A1 US 9005245 W US9005245 W US 9005245W WO 9105045 A1 WO9105045 A1 WO 9105045A1
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WO
WIPO (PCT)
Prior art keywords
cdna sequence
sequence
lactoferrin
porcine
cdna
Prior art date
Application number
PCT/US1990/005245
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English (en)
Inventor
Denis R. Headon
Orla M. Conneely
Bert W. O'malley
Original Assignee
Granada Biosciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Granada Biosciences, Inc. filed Critical Granada Biosciences, Inc.
Publication of WO1991005045A1 publication Critical patent/WO1991005045A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • Lactoferrin is an iron-binding glycoprotein found in milk and other secretions and body fluids. It is one of a number of iron binding proteins, sometimes referred to as transferrins, and is involved in iron binding and delivery in mammals.
  • Lactoferrin has been implicated as a factor in resis ⁇ tance against enteritis infections in suckled newborns. These bacteriocidal/bacteriostatic actions are considered to be due, in part, to the iron binding properties of the protein. Lactoferrin decreases the iron availability to iron requiring microorganisms and thereby interferes with their growth and reproduction. Lactoferrin is also con ⁇ sidered to have antiviral properties and other potential therapeutic applications.
  • hLF Human lactoferrin
  • Bovine lactoferrin has been isolated and is used as a milk additive.
  • a protein Identified as mouse lacto ⁇ transferrin has been isolated.
  • the cDNA sequence and deduced amino acids have been reported. Pentecost and Teng, "Lactotransferrin Is The Major Estrogen Inducible Protein of Mouse Uterine Secretions", 262 J. Biol. Che . , pp. 10134- 10139 (1987).
  • Pig lactoferrin has not been sequenced for amino acids or nucleotides. Piglets suffer from anemia; there- fore, an enhanced iron delivery system would be valuable. Also, therapeutic uses of lactoferrins have been reported- which would benefit humans and animals.
  • the invention is the cDNA sequence for the partial porcine or pig lactoferrin (pLF) protein which includes two iron binding sites.
  • the porcine cDNA sequence lacks about 105 nucleotides from the coding sequence in the second iron binding domain of the mature protein.
  • the cDNA sequence does not code for the entire protein but for a substantial part of the protein.
  • Part of the cDNA sequence may be used to code a polypeptide with the entire iron binding domain closest to the N-terminal end of the protein.
  • a complete cDNA sequence includes nucleotides coding for a secretion signal peptide which enables the mature pLF to cross the cell membrane.
  • the nucleotide sequence for the porcine cDNA coding the secretion signal peptide is incom ⁇ plete.
  • the signal peptide for hLF can be used with the porcine nucleotide sequence.
  • the present invention of the cDNA sequence can be used to prepare a recombinant porcine lactoferrin product, thus making available a source for therapeutic and nutritional applications.
  • the cDNA sequence of this invention can be used in an appropriate cloning vehicle to replicate the cDNA sequence.
  • part or all of the cDNA can be incorporated into a vector system for expression. This invention is not limited to any particular uses of the cDNA.
  • the inclusion of a substantial part of the lactoferrin gene with expres ⁇ sion should improve an animal's disease resistance to bacterial and viral infection.
  • the tissue specific expres ⁇ sion of lactoferrin in mammary glands would impart the bacteriocidal and viracidal benefit of the expressed gene to young feeding on the milk and would provide a production means for the secreted protein for therapeutic use.
  • the gene can be placed in the appropriate cloning vector for the production of a synthetic pLF product.
  • the pLF polypeptide produced by recombinant methods can be used in a variety of products including feed, therapeutic addi ⁇ tives to enhance iron transport and delivery and for the viracidal and bacteriocidal qualities, additives for eye- drops, contact lens and other eye care solutions, topical skin care products, ear drops, mouthwashes, chewing gum and toothpaste.
  • the recombinant pLF polypeptide product would provide a safe, naturally occurring product which can be topically applied as well as ingested safely.
  • Fig. 1 is the number cDNA sequence from 5' to 3' end with the partial porcine signal peptide sequence and the partial mature porcine lactoferrin protein sequence and untranslated regions with corresponding deduced amino acids.
  • Fig. 2 is a cDNA sequence from 5' to 3' end and corre ⁇ sponding amino acids starting with part of the untranslated hLF, hLF signal peptide sequence, the mature porcine lacto ⁇ ferrin nucleotide sequence and the untranslated pLF cDNA at the 3' end.
  • the cDNA sequence for the porcine lactoferrin gene including iron-binding sites for two iron binding domains is shown in Fig. 1.
  • the sequence in Fig. 1 starting at the 5' end includes an untranslated region of undetermined length which is not sequenced shown as "NNNNNNNNNN”.
  • the next sequence from the 5' end is a signal oeptide for secretion for pLF lacking two amino acids.
  • the signal peptide is not part of the mature pLF but is necessary for effective secretion of the protein.
  • the following portion of the cDNA sequence in Fig. 1 starting with the nucleotides coding for amino acid number 20 alanine is the full cDNA sequence for the first iron binding domain of pLF.
  • the iron binding domains require an adequate number of amino acids in sequence for three dimensional confirmation to align the iron molecule with the binding sites.
  • the iron binding sites closest to the N-terminal end of pLF are disclosed.
  • the full functional domain is shown in Fig. 1.
  • a polypeptide product may be prepared using the sequence shown in Fig. 1 for the complete iron binding N-terminal end of the protein.
  • amino acids asparagine, tyrosine, tyrosine and histidine are iron binding sites of the first domain closest to the N-terminal end of the protein.
  • the cDNA sequence has been determined up to the corresponding nucleotide for the amino acid asparagine, number 463. Thereafter, about 105 nucleotides have not been determined.
  • nucleotide number and amino acid numbers are assigned for convenience of reference.
  • the omitted nucleotide bases are represented as repeated "N's" and are in the region of the second iron binding domain.
  • the iron binding sites for the domain nearest the carboxy terminus of the protein are identified for the second domain as amino acids asparagine, tyrosine, tyrosine and histidine, numbers 409, 448, 540 and 609, respectively.
  • glutamine numbered as amino acid 499 of Fig. 1, the corresponding cDNA sequence for pLF is complete for the rest of the mature protein including the carboxy terminal end of the protein.
  • the cDNA sequence continues to the '3 end with an untranslated region starting at nucleotide number 2068.
  • the pLF cDNA and amino acid sequence to the nearly complete signal peptide, mature protein and untranslated region is shown in Fig. 1.
  • the pLF cDNA disclosed does not code the entire protein.
  • a polypeptide coded by the cDNA of the invention can include only the functional domain around the iron binding sites useful in iron delivery or any other useful amino acid sequence. It is not the intention to limit the invention to the entire cDNA sequence shown in Fig. 1.
  • Any useful pLF polypeptide product coded by all or part of the cDNA sequence is included in this invention.
  • any portion of the cDNA sequence used with an expression vector to code a useful polypeptide is part of this invention.
  • One or more of the cDNA sequences for the functional iron binding domains may be placed in a vector for expression.
  • the cDNA sequence disclosed is a map from which to select the nucleotide sequence to code for the desired pLF polypeptide product. There are no limitations intended for the use of the cDNA sequence to produce a useful polypeptide.
  • the sequence in Fig. 2 is an example of the use of cDNA sequence coding the mature pLF from Fig. 1 in combination with the entire sequence coding for the human secretion signal peptide for hLF.
  • the mature pLF cDNA sequence or parts thereof can be joined with the hLF signal peptide to create a new polypeptide of this invention.
  • a short portion of untranslated hLF from the 5' end is shown at the beginning of the cDNA sequence.
  • the numbering of the nucleotide bases starts at the 5" untranslated end.
  • the sequence in Fig. 2 ends with an untranslated pLF cDNA at the 3' end. It is not necessary to use the untranslated cDNA at either the 3' or the 5' end to prepare a polypeptide of this invention.
  • the human signal portion will effect secretion of the mature porcine lactoferrin polypeptide.
  • the human signal peptide and corresponding cDNA sequence is:
  • the nucleotide sequence analysis was performed on cDNA isolated from a porcine mammary cDNA library.
  • the library yielded two cDNA clones with sequences for analysis.
  • One clone contained a sequence 1391 base pairs (bp) in length and included all but two amino acids of pLF secretion signal peptide sequence.
  • the 1391 bp clone contained the first 463 amino acids of mature pLF and encoded the complete N-terminal iron binding domain.
  • the second cDNA sequence was 801 base pairs in length. It encoded the carboxy-terminal 162 amino acids of mature pLF followed by 238 nucleotides of the 3' untranslated region.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Une séquence d'ADNc pour une protéine de lactoférine porcine (pLF) et une séquence d'acides aminés correspondante ont été découvertes. On peut utiliser la séquence d'ADNc pour préparer des produits de recombinaison de lactoférine de porc en vue d'applications thérapeutiques et alimentaires. Des régions de la séquence d'ADNc comme des sites de liaison Fe peuvent être utilisées pour réaliser un produit de polypeptide pLF.
PCT/US1990/005245 1989-09-28 1990-09-14 SEQUENCE D'ADNc ET D'ACIDE AMINE DE LACTOFERINE PORCINE WO1991005045A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US41388089A 1989-09-28 1989-09-28
US413,880 1989-09-28

Publications (1)

Publication Number Publication Date
WO1991005045A1 true WO1991005045A1 (fr) 1991-04-18

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PCT/US1990/005245 WO1991005045A1 (fr) 1989-09-28 1990-09-14 SEQUENCE D'ADNc ET D'ACIDE AMINE DE LACTOFERINE PORCINE

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AU (1) AU6406590A (fr)
CA (1) CA2025530A1 (fr)
IE (1) IE903471A1 (fr)
IL (1) IL95697A0 (fr)
WO (1) WO1991005045A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5565425A (en) * 1993-03-04 1996-10-15 Snow Brand Milk Products Co., Ltd Viral infection and proliferation inhibitors
US5571691A (en) * 1989-05-05 1996-11-05 Baylor College Of Medicine Production of recombinant lactoferrin and lactoferrin polypeptides using CDNA sequences in various organisms
US5571697A (en) * 1989-05-05 1996-11-05 Baylor College Of Medicine Texas Medical Center Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in Aspergillus
US5766939A (en) * 1989-05-05 1998-06-16 Baylor College Of Medicine Production of recombinant lactoferrin and lactoferrin polypeptides using CDNA sequences in various organisms
US5849881A (en) * 1989-05-05 1998-12-15 Baylor College Medicine Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms
US6100054A (en) * 1989-05-05 2000-08-08 Baylor College Of Medicine Production for recombinant lactoferrin and lactoferrin polypeptides using DNA sequences in various organisms
US6111081A (en) * 1996-05-31 2000-08-29 Baylor College Of Medicine Lactoferrin variants and uses thereof
US6228614B1 (en) 1989-05-05 2001-05-08 Baylor College Of Medicine Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHIMICA ET BIOPHYSICA ACTA vol. 999, no. 3, 21 December 1989, ELSEVIER SCIENCE PUBLISHERS B.V. pages 323 - 329; HUTCHENS, T.W. et al.: "Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence." SA 40297 030see the whole document *
BLOOD vol. 79, no. 4, 1987, pages 989 - 993; RADO, T.A. et al.: "Isolation of Lactoferrin cDNA from a human myeloid library and expressionof mRNA during normal and leukemic myelopoiesis." see the whole document *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5571691A (en) * 1989-05-05 1996-11-05 Baylor College Of Medicine Production of recombinant lactoferrin and lactoferrin polypeptides using CDNA sequences in various organisms
US5571697A (en) * 1989-05-05 1996-11-05 Baylor College Of Medicine Texas Medical Center Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in Aspergillus
US5766939A (en) * 1989-05-05 1998-06-16 Baylor College Of Medicine Production of recombinant lactoferrin and lactoferrin polypeptides using CDNA sequences in various organisms
US5849881A (en) * 1989-05-05 1998-12-15 Baylor College Medicine Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms
US5955316A (en) * 1989-05-05 1999-09-21 Agennix, Inc. Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in aspergillus
US6080559A (en) * 1989-05-05 2000-06-27 Agennix, Inc. Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in Aspergillus
US6100054A (en) * 1989-05-05 2000-08-08 Baylor College Of Medicine Production for recombinant lactoferrin and lactoferrin polypeptides using DNA sequences in various organisms
US6228614B1 (en) 1989-05-05 2001-05-08 Baylor College Of Medicine Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms
US5565425A (en) * 1993-03-04 1996-10-15 Snow Brand Milk Products Co., Ltd Viral infection and proliferation inhibitors
EP0871724A4 (fr) * 1994-11-02 1998-10-21
EP0871724A1 (fr) * 1994-11-02 1998-10-21 Agennix, Inc. Expression de lactoferrines et de fragments polypeptidiques de lactoferrines recombines traites, a partir d'un produit de fusion, dans aspergillus
US6111081A (en) * 1996-05-31 2000-08-29 Baylor College Of Medicine Lactoferrin variants and uses thereof

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Publication number Publication date
AU6406590A (en) 1991-04-28
CA2025530A1 (fr) 1991-03-29
IE903471A1 (en) 1991-04-10
IL95697A0 (en) 1991-06-30

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