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Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins

a technology of plant gums and glycoproteins, which is applied in the field of synthetic genes for plant gums and other hydroxyproline-rich glycoproteins, can solve the problems of reducing the number of cells receiving, affecting the quality of plant gums, so as to achieve the effect of maximizing the number of cells

Inactive Publication Date: 2004-01-15
OHIO UNIV TECH TRANSFER OFFICE TECH & ENTERPRISE BUILDING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention relates to the field of plant gums and other hydroxyproline-rich glycoproteins (HRGPs). These proteins are ubiquitous in the plant kingdom and are involved in various aspects of plant growth and development. The invention involves the design of polypeptides that mimic the structure and function of HRGPs, as well as the production of synthetic genes and the construction of expression vectors for the polypeptides. The invention also includes the use of O-glycosylation codes to create the unique molecular surface topography of HRGPs. The technical effects of the invention include the creation of polypeptides that can be used to improve plant growth and development, as well as the production of synthetic genes and the construction of expression vectors for the polypeptides."

Problems solved by technology

It is a tedious process involving piercing and stripping the bark of the trees, then returning later to gather the dried tear drop shaped, spherical balls that form in response to mechanical wounding.
However, this production depends on the environmental and political stability of the region producing the gum.
Again in 1985, drought brought about shortages of the gum, resulting in a 600% price increase.
Second, additives have been investigated to supplement inferior gum arabic.
The effort to find other gums in other regions of the world has met with some limited success.
However, this approach has limitations.
Unfortunately, conditions have not been found which lead to the expression of gum arabic in culture. A. Mollard and J -P. Joseleau, "Acacia senegal cells cultured in suspension secrete a hydroxyproline-deficient arabinogalactan-protein" Plant Physiol. Biochem. 32:703 (1994).

Method used

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  • Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins
  • Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins
  • Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins

Examples

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example 1

Determination of the Peptide Sequence of Acacia Gum Arabic Glycoproteins

[0182] In this example, GAGP (SEQ ID NO:15) was isolated and (by using chymotrypsin) the deglycosylated polypeptide backbone was prepared. Although GAGP does not contain the usual chymotryptic cleavage sites, it does contain leucyl and histidyl residues which are occasionally cleaved. Chymotrypsin cleaved sufficient of these "occasionally cleaved" sites to produce a peptide map of closely related peptides.

[0183] Purification and Deglycosylation of GAGP (SEQ ID NO:15). GAGP was isolated via preparative Superose-6 gel filtration. Anhydrous hydrogen fluoride deglycosylated it (20 mg powder / mL HF at 4.degree. C., repeating the procedure twice to ensure complete deglycosylation), yielding dGAGP which gave a single symmetrical peak (data not shown) after re-chromatography on Superose-6. Further purification of dGAGP by reverse phase chromatography also gave a single major peak, showing a highly biased but constant ami...

example 2

Construction of Synthetic HRGP Gene Cassettes

[0186] Synthetic gene cassettes encoding contiguous and noncontiguous Hyp modules are constructed using partially overlapping sets consisting of oligonucleotide pairs, "internal repeat pairs" and "external 3'- and 5'-linker pairs" respectively, all with complementary "sticky" ends. The design strategy for the repetitive HRGP modules combines proven approaches described earlier for the production in E. coli of novel repetitive polypeptide polymers (McGrath et al. [1990] Biotechnol. Prog. 6:188), of a repetitious synthetic analog of the bioadhesive precursor protein of the mussel Mytilus edulis, of a repetitive spider silk protein (Lewis et al. [1996] Protein Express. Purif. 7:400), and of a highly repetitive elastin-like polymer in tobacco [Zhang, X., Urry, D. W., and Daniell, H. "Expression of an environmentally friendly synthetic protein-based polymer gene in transgenic tobacco plants," Plant Cell Reports, 16: 174 (1996)].

[0187] The basi...

example 3

Isolation of Tomato P1 Extensin cDNA Clones

[0211] In order to obtain the tomato P1 extensin signal sequence (i.e., signal peptide), P1 extensin cDNA clones were isolated using oligonucleotides designed after the P1-unique protein sequence (SEQ ID NO:51): Val-Lys-Pro-Tyr-His-Pro-Thr-Hyp-Val-Tyr-Lys. When present at the N-terminus of a protein sequence, the P1 extensin signal sequence directs the nascent peptide chain to the ER.

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Abstract

A new approach in the field of plant gums is described which presents a new solution to the production of hydroxyproline(Hyp)-rich glycoproteins (HRGPs), repetitive proline-rich proteins (RPRPs) and arabinogalactan-proteins (AGPs). The expression of synthetic genes designed from repetitive peptide sequences of such glycoproteins, including the peptide sequences of gum arabic glycoprotein (GAGP), is taught in host cells, including plant host cells.

Description

[0001] The present invention relates generally to the field of plant gums and other hydroxyproline-rich glycoproteins, and in particular, to the expression of synthetic genes designed from repetitive peptide sequences.[0002] Gummosis is a common wound response that results in the exudation of a gum sealant at the site of cracks in bark. A. M. Stephen et al., "Exudate Gums", Methods Plant Biochem. (1990). Generally the exudate is a composite of polysaccharides and glycoproteins structurally related to cell wall components such as galactans [G. O. Aspinall, "Plant Gums", The Carbohydrates 2B:522536 (1970)] and hydroxyproline-rich glycoproteins [Anderson and McDougall, "The chemical characterization of the gum exudates from eight Australian Acacia species of the series Phyllodineae." Food Hydrocolloids, 2: 329 (1988)].[0003] Gum arabic is probably the best characterized of these exudates (although it has been largely refractory to chemical analysis). It is a natural plant exudate secre...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09A61K38/17C07K14/00C07K14/415C07K19/00C12N5/10C12N15/29C12N15/82C12P21/02
CPCC07K14/415C12N15/8242C12N15/8241C07K2319/00
Inventor KIELISZEWSKI, MARCIA J.
Owner OHIO UNIV TECH TRANSFER OFFICE TECH & ENTERPRISE BUILDING
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