EP0964918A2 - Reversibly inactive synthetic beta-galactosidase - Google Patents

Reversibly inactive synthetic beta-galactosidase

Info

Publication number
EP0964918A2
EP0964918A2 EP97953997A EP97953997A EP0964918A2 EP 0964918 A2 EP0964918 A2 EP 0964918A2 EP 97953997 A EP97953997 A EP 97953997A EP 97953997 A EP97953997 A EP 97953997A EP 0964918 A2 EP0964918 A2 EP 0964918A2
Authority
EP
European Patent Office
Prior art keywords
enzyme
galactosidase
milk
synthetic
dna
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP97953997A
Other languages
German (de)
English (en)
French (fr)
Inventor
Costas N. Karatzas
Jeffrey D. Turner
Mahmoud Eino
John J. Kabel
Gerald F. Amantea
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nexia Biotechnologies Inc
Original Assignee
Nexia Biotechnologies 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 Nexia Biotechnologies Inc filed Critical Nexia Biotechnologies Inc
Publication of EP0964918A2 publication Critical patent/EP0964918A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • Lactose is one of the main milk components and it is found exclusively in dairy
  • Lactose cannot be absorbed directly by the intestine. Rather, it must be cleaved into its
  • lactase-phlorizin hydrolase lactase-phlorizin hydrolase
  • lactose intolerance is characterized by a number of gastrointestinal disorders when the
  • lactose malabsorption and milk intolerance affects the majority of the world's population.
  • the present invention features the engineering of a ⁇ -galactosidase protein
  • pancreatic secretions ⁇ -galactosidase genes are available that encode enzymes which
  • ⁇ -galactosidase is 3-4, that of Aspergillus oryzae is about 5.0, while that o ⁇ K. lactis is
  • the time parameter in the stomach is governed by the rate of flow of the chyme
  • the volume of the meal is the predominant regulator of
  • the invention provides a synthetic ⁇ -galactosidase enzyme which differs from
  • cleavage site preferably an enzyme-cleavable peptide bond
  • regulatory domain is to reversibly inactivate/suppress the hydrolyzing activity of
  • the cleaving enzyme is one which is present preferably in the human
  • the cleavage site should be one
  • the invention also features DNA molecules encoding the synthetic enzymes of the
  • the invention also features transgenic mammals whose mammary glands express the
  • DNA molecule encoding the synthetic enzyme includes a
  • mammary-specific promoter controlling transcription of the DNA encoding the enzyme
  • Fig. 1 is a diagrammatic representation of the construction of a plasmid used as the
  • Fig. 2 is a diagrammatic representation of the construction of a vector containing A. niger ⁇ -galactosidase-encoding DNA under the control of the ⁇ -Gla ( ⁇ -glucoamylase)
  • Fig. 3 is a diagrammatic representation of the construction of a vector containing
  • DNA encoding A. niger ⁇ -galactosidase fused to a bovine lactoferrin tail.
  • Fig. 4 is a diagrammatic representation of the construction of a vector containing
  • DNA encoding A. niger ⁇ -galactosidase fused to a polyamino acid tail-encoding sequence.
  • Fig. 5 is a diagrammatic representation of the construction of a vector containing a
  • FIG. 6 is a diagrammatic representation of an expression cassette in which the A. niger
  • ⁇ -galactosidase gene is fused to A. niger ⁇ -galactosidase cDNA (tail) through a pepsin
  • Fig. 7 is a diagrammatic representation of a vector in which the 3' end of the
  • ⁇ -galactosidase gene is modified to encode a protein with a cleavage site recognizable by
  • Fig. 8 is a diagrammatic representation of the fusion of the modified ⁇ -galactosidase
  • Fig. 9 is a diagrammatic representation of the strategy of the construction of a vector
  • genomic A. niger ⁇ -galactosidase-encoding DNA fused through a pepsin site to
  • A. niger ⁇ -galactosidase cDNA (tail).
  • Fig. 10 is a diagrammatic representation of the overall strategy for constructing a
  • the enzyme to be used must be acid resistant and resist denaturation within the
  • stomach from pH 1.0 (fasting state) to 5.0.
  • the enzyme must retain activity in the presence of
  • proteolytic enzymes pepsin in the stomach and the pancreatic proteases in the intestine
  • the ⁇ -galactosidase of A. niger has a pH optimum in the range of 3-5, which makes it
  • This enzyme has relatively high
  • the ⁇ -galactosidase of A. niger is a suitable ⁇ -galactosidase of A. niger.
  • the pro-enzyme is completely inactive. Following secretion from the cell, the pro-enzyme is
  • Gastrointestinal protease structure and function are well understood.
  • the target is Gastrointestinal protease structure and function.
  • the target is Gastrointestinal protease structure and function.
  • the target is Gastrointestinal protease structure and function.
  • the target is Gastrointestinal protease structure and function.
  • Pepsin is found in significant quantities within the gastrointestinal system. Its presence is a
  • construct involved a bovine casein promoter driving the expression of bovine chymosin
  • Chymosin was synthesized as a prochymosin which is
  • Fusion-tail systems have been developed using
  • polyamino acid tails have been generated in order to facilitate their purification (Niederauer
  • ⁇ -galactosidase for example, through an enzyme recognition/cleavage recognition site
  • the cleavage sequence is engineered for recognition by a proteolytic enzyme.
  • constructs include a promoter region
  • antibiotic resistant genes for example amp
  • sequences depends on the host organism to be used as the expression system. These components are addressed below:
  • the signal peptide can be one from the endogenous ⁇ -galactosidase gene if it is a
  • prolactase is to be produced (i.e. from the A. niger glucoamylase gene). If the prolactase is to be produced
  • mammary epithelial cells into milk Such systems are described, e.g., in Gordon et al., 1987,
  • the ⁇ -galactosidase e.g., from A. niger or A. oryzae, has desirable functional
  • the fused gene can be engineered to include a cleavage site
  • the engineered tail will render the ⁇ -galactosidase molecule reversibly inactive due to
  • the enzyme may also serve as an affinity handle during purification of the enzyme from culture broth.
  • the tail domain may be added at the 3'-, 5'- or both ends of the gene whose activity is
  • Proteins of known length and function which are normal constituents of milk such as caseins, lactoferrin or lactalbumin can be used as tails.
  • tails Proteins of known length and function which are normal constituents of milk such as caseins, lactoferrin or lactalbumin can be used as tails.
  • caseins lactoferrin or lactalbumin
  • lactoferrin or lactalbumin can be used as tails.
  • polyaminoacid tails can be used that add either a positive or negative charge to the
  • Truncation can be either at the amino or carboxyl
  • prolactase molecules (with the tail part having a series of deletions) are provided.
  • the antibodies used can recognize either the ⁇ -galactosidase or the
  • a preferred fungal host is for example the one
  • the cleavage sequence is engineered to act as a recognition site for the specific
  • Table 1 provides a partial list of such proteolytic enzymes and their target
  • cleavage sites can be added for a specific application. For example, activation of the prolactase in the gastric environment would require a site that is recognized
  • regulatory domain can generate a fusion molecule that could be added in milk during the
  • the tail is removed, and the ⁇ -galactosidase is released, thereby hydrolyzing the lactose
  • Prokaryotic fermentation typically utilizes Escherichia coli
  • Saccharomyces cerevisiae (Goff et al, 1984) is routinely used. Fungal fermentation also
  • the expression vector was derived from the pGla expression vector kindly provided
  • step 1) was first digested with Sal restriction enzyme (Promega, Madison, WI, USA) and
  • kb band containing the pUC18 backbone was purified using the QiaxII kit. The two purified
  • ⁇ -galactosidase cDNA or structural gene was restricted with Xhol which excises the A. niger
  • bovine lactoferrin (BLF) cDNA was cloned from mRNA isolated from bovine
  • the PCR was performed in a final volume of 100 ⁇ l reaction using
  • the underlined sequences represent overlapping sequences so that the amplified 3' sequence
  • ⁇ -gal genomic DNA was fused with the BLF sequence using PCR without primers.
  • the fusion product was then subcloned into a pGEMT vector that allows direct subcloning of PCR products. The resulting
  • vector pGEMT/bgblfPCR was restricted with Nael and SpHI and ligated into the Nael, SpHI
  • GTF7 genomic ⁇ -galactosidase gene
  • Tails are to be fused to the 3'-end of the ⁇ -galactosidase
  • prolactase containing bovine -lactalbumin (used as the regulatory domain fused at the 3'-end of the ⁇ -galactosidase gene with or without a pepsin recognition site
  • Bovine ⁇ -lactalbumin was cloned by RT-PCR. Total RNA was first extracted from
  • PCR fragment was digested with Agel and Notl and ligated to the Agel, Notl sites of the
  • the resulting plasmid contains the a-lac gene fused at the
  • the objective was to construct an expression cassette, pGgbpb (Fig. 6), based on the
  • the glucoamylase promoter (Element 1), full length genomic ⁇ -galactosidase gene
  • the starting vector is pGTF7 (Fig. 9). This vector contains pUC18 backbone, Element
  • pSPanb Fig. 8 which are pSP72 vectors containing genomic and cDNA ⁇ -galactosidase
  • the construction replaced Element 2 of the plasmid pGTF7 (Fig. 9) with a DNA
  • the DNA template is the plasmid pSPganb, which contains the A. niger ⁇ -galactosidase
  • the 5' primer was 5'-CAAGAACGGCATCTGGTCAG-3', and the 3' primer
  • the pepsin recognition site (Element 3) and suitable cloning sites are (Agel and BssHII)
  • Suitable cloning sites are designed to fuse the ⁇ -galactosidase cDNA sequences at the
  • the DNA template for PCR was plasmid pSPanb
  • the 5' primer is 5'-ATTAGCGCGCGAACTGTTGCAGAAATACGTC-3*, and
  • deoxyribonucleotide(dNTP) 0.25 uM of each primer and the thermo DNA polymerase buffer.
  • the reaction was carried out in MiniCycler (MJ Research Inc.) using the following
  • plasmid pSPgbpb (Fig.8) which contains Element 2, Element 3 and Element 4.
  • cDNA served as a template for the cloning of the ⁇ -casein gene.
  • the second 3' end primer contains a unique Not I site to facilitate
  • the PCR product (.56kb) was restricted with bpnl/Notl (step 2) and subcloned into
  • pepsin will be cleaving the pepsin recognition site (between the b-gal gene and
  • Lactose hydrolysis is measured by a
  • the model system developed is based on conditions prevailing in the stomach. Gastric
  • juice is a simple fluid containing 150 meq/ml of hydrogen (pH of 1.5) and 0.5 to 1 mg/ml of
  • the pure juice at high rates of secretion can have a pH below 1 , but upon
  • pH has been estimated to range between 1.5 and 2.5.
  • a range of pepsin concentration from 0.1 to 10 mg/ml is used in order to take into account the great variability of pepsin found in
  • the activity of the prolactase is tested using 400 ul of milk by the addition 3 different
  • hydrochloric acid e.g., hydrochloric acid, pepsin and prolactase or lactase (used as the positive control).
  • Varying the quantity of hydrochloric acid added allow us to cover a range of pH from about
  • Lactose concentrations are determined by HPLC analysis.
  • pepsin stock solution (100 mg/ml).
  • prochymosin derivatives containing extension of various length of the pro-part.
  • subtilis FEMS Microbiol. Lett. 61:243-249.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Dairy Products (AREA)
EP97953997A 1996-12-31 1997-12-29 Reversibly inactive synthetic beta-galactosidase Withdrawn EP0964918A2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US775842 1985-09-13
US77584296A 1996-12-31 1996-12-31
PCT/IB1997/001658 WO1998029536A2 (en) 1996-12-31 1997-12-29 Reversibly inactive synthetic beta-galactosidase

Publications (1)

Publication Number Publication Date
EP0964918A2 true EP0964918A2 (en) 1999-12-22

Family

ID=25105684

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97953997A Withdrawn EP0964918A2 (en) 1996-12-31 1997-12-29 Reversibly inactive synthetic beta-galactosidase

Country Status (4)

Country Link
EP (1) EP0964918A2 (ja)
JP (1) JP2001506136A (ja)
AU (1) AU5775798A (ja)
WO (1) WO1998029536A2 (ja)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120230973A1 (en) * 2009-09-22 2012-09-13 Amano Enzyme Inc. Lactase preparation
CN107857801B (zh) * 2017-10-23 2020-09-22 华南理工大学 一种可用于提高分泌效率的信号肽及其应用
CN107698666A (zh) * 2017-10-23 2018-02-16 华南理工大学 一种可有效提高分泌的信号肽及其应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US551459A (en) * 1895-12-17 Counter-fixture
EP0832981A1 (en) * 1987-02-17 1998-04-01 Pharming B.V. DNA sequences to target proteins to the mammary gland for efficient secretion
GB8905674D0 (en) * 1989-03-13 1989-04-26 Imperial College Dna construct and modified yeast
JP3662948B2 (ja) * 1992-12-09 2005-06-22 ニユー・イングランド・バイオレイブス・インコーポレイテツド 改質タンパク質及びその製造法
US5780009A (en) * 1995-01-20 1998-07-14 Nexia Biotechnologies, Inc. Direct gene transfer into the ruminant mammary gland
US5821350A (en) * 1995-11-01 1998-10-13 Nexia Biotechnologies, Inc. Aspergillus niger beta-galactosidase gene

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9829536A3 *

Also Published As

Publication number Publication date
WO1998029536A3 (en) 1998-09-11
AU5775798A (en) 1998-07-31
JP2001506136A (ja) 2001-05-15
WO1998029536A2 (en) 1998-07-09

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