Unlock instant, AI-driven research and patent intelligence for your innovation.

Transgenic plants expressing cobalamin binding proteins

a technology of cobalamin and binding proteins, which is applied in the field of transgenic plant production of recombinant proteins, can solve the problems of complex isolation of pure tc and if from natural sources, high cost of expression systems, and risk of human disease transmission from donor to patient, and achieves the effect of easy scaling up and low cos

Inactive Publication Date: 2015-06-11
XERAGENX
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a method for producing a protein called cobalamin binding protein (CBP) in plants. By introducing a foreign nucleic acid into the mitochondria or chloroplasts of a plant, CBP can be expressed in a way that avoids contamination of other plants in the environment. The use of plants for large-scale production of CBP is an efficient and cost-effective alternative to traditional methods. Additionally, the invention describes a mathematical algorithm for comparing plant sequences.

Problems solved by technology

Isolation of pure TC and IF from natural sources is also complicated by the presence of relatively large amounts of HC in these sources.
These expression systems are expensive to use and they deliver a mixture of holo- and apo-forms of the CBP.
Therefore, there is a risk for transmission of human diseases from the donor to the patient.
Similarly, the use of IF isolated from pig or recombinant human IF isolated from the yeast expression media which contains an enzymatic hydrolysate of meat, may cause a risk for transmission of animal diseases to the patient.
Isolation of IF from other sources, i. e. gastric juice results in a preparation contaminated with some HC since both CBPs are present in gastric juice.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Transgenic plants expressing cobalamin binding proteins
  • Transgenic plants expressing cobalamin binding proteins
  • Transgenic plants expressing cobalamin binding proteins

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0060]As shown in FIG. 1, the extensin signal peptide encoding nucleotide sequence from Arabidopsis thaliana was fused to the nucleotides encoding mature human intrinsic factor (FIG. 2). This construct was inserted in the plant transformation vector CRC179.

Construction of the CRC-179 vector

[0061]The vector pPZP 211 (Hajdukiewicz, P; Svab, Z; & Maliga, P. 1994 Plant Mol. Biol. 25, 989-994) was digested with EcoRI and KpnI and a pAnos sequence was released from pGPTV KAN (Becker, D; Kemper, E; Schell, J & Masterson, R. 1992 Plant Mol Biol 20, 1195-1197) by the same set of enzymes and cloned into the pPZP 211 vector. The resulting vector was digested with PstI and KpnI and blunt-ended. A blunt-ended EcoRI and HindIII fragment containing the 35S CaMV promoter from the vector described in Jefferson, R A; Kavanagh, T A & Bevan, M W. 1987 EMBO J. 6,3901-3907 was cloned into the blunt-ended vector. This vector was named CRC-179.

[0062]The bacteria Agrobacterium tumefaciens was transformed wi...

example 2

[0076]Arabidopsis thaliana was transformed with a vector construct which contained a nucleotide sequence encoding the signal peptide from the Phaseolus vulgaris chitinase CH5B fused to the nucleotide sequence encoding mature human intrinsic factor (FIG. 3). This construct was used to generate transgenic Arabidopsis thaliana plants. These plants were shown to contain CBC at the same level as most of the extensin-IF plants showing that the choice of signal peptide for intrinsic factor expression is not restricted to one sequence.

example 3

[0077]Arabidopsis thaliana was transformed with a vector construct which contained a nucleotide sequence encoding the signal peptide from the Nicotiana tabacum glucan beta-1,3-glucanase fused to the nucleotide sequence for mature human intrinsic factor (FIG. 4). This construct was used to generate transgenic Arabidopsis thaliana plants. These plants were shown to contain CBC at the same level as most of the extensin-IF plants showing that the choice of signal peptide for intrinsic factor expression is not restricted to one sequence.

Conclusions from IF-Plants in Examples 1-3

[0078]As far as we have tested the recombinant human IF from plants it behaves as natural human gastric IF concerning its mature N-terminus, recognition by anti-IF antibodies, binding of cobalamin, lack of cobinamide binding, binding to the intestinal receptor, and presence of carbohydrates. In contrast to gastric juice where IF is present together with another CBP, haptocorrin, and to some extent cobalamin from t...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
pHaaaaaaaaaa
humidityaaaaaaaaaa
molecular weightaaaaaaaaaa
Login to View More

Abstract

The present invention relates to the use of transgenic plants for the expression of vitamin B12 (cobalamin) binding proteins. Plant cells are transformed with nucleotide sequences adapted for expression and secretion of vitamin B12 binding proteins. The present invention also relates to the use of recombinant vitamin B12 binding proteins from plants in analytical tests and treatment of vitamin B12 deficiency. Also disclosed is a method for purification of recombinant vitamin B12 binding proteins.

Description

FIELD OF THE INVENTION[0001]The present invention generally relates to transgenic plant production of recombinant proteins which are able to bind cobalamin. The recombinant proteins may be used 1) prophylactically and / or therapeutically to treat cobalamin deficiency and 2) analytically.BACKGROUND OF THE INVENTION[0002]Two groups of proteins are characterized by specific binding to vitamin B12, (cobalamin), hereafter called cobalamin binding proteins (CBP). One group consists of proteins involved in the assimilation of cobalamin and the other group is involved in the metabolism and utilization of cobalamin and includes the enzymes using cobalamin as a cofactor. Human assimilation of dietary cobalamin (Cbl) is a complex process with three successive Cbl-transporters involved: haptocorrin (HC), intrinsic factor (IF) and transcobalamin (TC) (1-4). These proteins strongly bind to Cbl which is synthesized by bacteria. The three CBPs provide uptake of the Cbl from the food to the body. IF ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): A61K47/48A61K31/7056A61K38/16C07K14/415A01H5/00A23L1/305A23L19/00A61K38/00A61P3/02C07K1/14C07K1/22C07K1/34C07K14/47C12N5/10C12N15/09C12N15/12C12N15/61C12N15/82G01N33/53G01N33/566G01N33/60G01N33/82
CPCA61K47/48107A61K38/168A61K31/7056C07K14/415A61K38/00C12N15/8257A61K47/551C07K14/47A61P3/02A01H1/00C12N5/0602C12N2500/38
Inventor BERGLUND, LARS ERIKPETERSEN, TORBEN EILEBAEKFEDOSOV, SERGEY NIKOLAEVICHNEXO, EBBALAURSEN, NIELS BECHJENSEN, ERIK OSTERGAARD
Owner XERAGENX