Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-11-17
2000-09-05
Myers, Carla J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 71, 435 698, 435 699, 43525411, 4352542, 536 231, 536 234, 536 237, 536 2374, 530350, C12P 2104, G01N 3353, C12N 114, C07H 2104
Patent
active
06114147&
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The pharmaceutical, the fine chemicals and the food industry need a number of compounds that have to be isolated from complex mixtures such as extracts of animal or plant tissue, or fermentation broth. Often these isolation processes determine the price of the product.
Conventional isolation processes are not very specific and during the isolation processes the compound to be isolated is diluted considerably with the consequence that expensive steps for removing water or other solvents have to be applied.
For the isolation of some specific compounds affinity techniques are used. The advantage of these techniques is that the compounds bind very specifically to a certain ligand. However these ligands are quite often very expensive.
To avoid spillage of these expensive ligands they can be linked to an insoluble Support. However, often linking the ligand is also expensive and, moreover, the functionality of the ligand is often affected negatively by such procedure.
So a need exists for developing cheap processes for preparing highly effective immobilized ligands.
SUMMARY OF THE INVENTION
The invention provides a method for immobilizing a binding protein capable of binding to a specific compound, comprising the use of recombinant DNA techniques for producing said binding protein or a functional part thereof still having said specific binding capability, said protein or said part thereof being linked to the outside of a host cell, whereby said binding protein or said part thereof is localized in the cell wall or at the exterior of the cell wall by allowing the host cell to produce and secrete a chimeric protein in which said binding protein or said functional part thereof is bound with its C-terminus to the N-terminus of an anchoring part of an anchoring protein capable of anchoring in the cell wall of the host cell, which anchoring part is derivable from the C-terminal part of said anchoring protein.
Preferably, the host is selected from Gram-positive bacteria and fungi, which have a cell wall at the outside of the host cell, in contrast to Gram-negative bacteria and cells of higher eukaryotes such as animal cells and plant cells, which have a membrane at the outside of their cells. Suitable Gram-positive bacteria comprise lactic acid bacteria and bacteria belonging to the genera Bacillus and Streptomyces. Suitable fungi comprise yeasts belonging to the genera Candida, Debaryomyces, Hansenula, Kluyveromyces, Pichia and Saccharomyces, and moulds belonging to the genera Aspergillus, Penicillium and Rhizopus. In this specification the group of fungi comprises the group of yeasts and the group of moulds, which are also known as lower eukaryotes. In contrast to the cells in plants and animals, the group of bacteria and lower eukaryotes are also indicated in this specification as microorganisms. The invention also provides a recombinant polynucleotide capable of being used in a method as described above, such polynucleotide comprising (i) a structural gene encoding a binding protein or a functional part thereof still having the specific binding capability, and (ii) at least part of a gene encoding an anchoring protein capable of anchoring in the cell wall of a Gram-positive bacterium or a fungus, said part of a gene encoding at least the anchoring part of said anchoring protein, which anchoring part is derivable from the C-terminal part of said anchoring protein. The anchoring protein can be selected from .alpha.-agglutinin, a-agglutinin FLO1, the Major Cell Wall Protein of a lower eukaryote, and proteinase of lactic acid bacteria. Preferably, such polynucleotide further comprises a nucleotide sequence encoding a signal peptide ensuring secretion of the expression product of the polynucleotide, which signal peptide can be derived from a protein selected from the .alpha.-mating factor of yeast, .alpha.-agglutinin of yeast, invertase of Saccharomyces, insulinase of Kluyveromyces, .alpha.-amylase of Bacillus, and proteinase of lactic acid bacteria. The polynucleotide can be operably linke
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patent: 5618290 (1997-04-01), Robinson et al.
Lipke et al., Mol. Cell, Biol. 9: 3155-3165, (Aug. 1989).
De Geus Pieter
Frenken Leon Gerardus J.
Klis Franciscus Maria
Toschka Holger York
Verrips Cornelis Theodorus
Johannsen Diana
Myers Carla J.
Unilever Patent Holdings
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