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Collagen producing plants and methods of generating and using same

a technology of collagen and plants, applied in the field of collagen producing plants and methods of generating, can solve the problems of poor proline hydroxylation of collagen, limited extensibility of collagen fibers, and great tensile strength of collagen fibers

Inactive Publication Date: 2012-11-08
COLLPLANT LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for producing collagen in a plant or isolated plant cell by expressing a collagen alpha chain and a P4H enzyme in a subcellular compartment devoid of endogenous P4H activity. The method can also involve expressing exogenous LH3 in the same compartment. The collagen alpha chain can be a signal peptide for targeting to an apoplast or vacuole. The invention also provides genetically modified plants or isolated plant cells capable of accumulating collagen with a hydroxylation pattern identical to that produced in human cells. The invention also provides a plant system comprising two genetically modified plants capable of accumulating collagen alpha chains.

Problems solved by technology

Type I collagen fibers provide great tensile strength and limited extensibility.
An attempt to produce human collagens that rely on the hydroxylation machinery naturally present in plants resulted in collagen that is poor in proline hydroxylation (Merle et al., 2002).

Method used

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  • Collagen producing plants and methods of generating and using same
  • Collagen producing plants and methods of generating and using same
  • Collagen producing plants and methods of generating and using same

Examples

Experimental program
Comparison scheme
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example 1

Constructs and Transformation Schemes

[0163]Constructions of expression cassettes and vectors used in this work are illustrated in FIG. 1a-d. All of the coding sequences in this work were optimized for expression in tobacco and chemically synthesized with desired flanking regions (SEQ ID NOs: 1, 4, 7, 12, 14, 16, 18, 20, 22). FIG. 1a—the synthetic genes coding for Col1 and Col2 (SEQ ID's 1, 4) fused either to the vacuolar signal or to the apoplast signal (encoded by SEQ ID NO: 7) or without signals were cloned in expression cassettes composed of a Chrysanthemum rbcS1 promoter and 5′ UTR (SEQ ID NO: 10) and a Chrysanthemum rbcS1 3′UTR and terminator (SEQ ID NO: 11). The complete expression cassettes were cloned in the multiple cloning site of the pBINPLUS plant transformation vector (van Engelen et al., 1995, Transgenic Res 4: 288-290). FIG. 1b—The synthetic genes coding for P4H beta-human, P4H alpha-human and P4H-plant (SEQ ID NOs: 12, 14 and 16) fused either to the vacuolar signal o...

example 2

Plant Collagen Expression

[0165]Synthetic polynucleotide sequences encoding the proteins listed in Table 1 below were designed and optimized for expression in tobacco plants.

TABLE 1List of expressed proteinsIncludedEncodedSwissProtAminoSplicingin SEQby SEQName:accessionacidsisoformDeletionsnameID NO.ID NO.Collagenp024521442OneER signalCol131alpha 1(I)versionchain[Precursor]Collagenp081231342OneER signalCol264alpha 2(I)Two changesversionchaindone in[Precursor]p08123:D549A andN249IProlyl 4-p07237487OneER signal,P4H1312hydroxylaseversionKDELbetaHumanbeta subunitProlyl 4-p13674517P13674-1ER signalP4H1514hydroxylasealphaHumanalpha-1subunitProlyl 4-No entry in252OneMitochon-P4Hplant1716hydroxylaseSwissprot.versiondrial signalPlantNCBIpredictedaccession:as: aa1-39gi:15227885Procollagenp13497866P13497-1ER signal,Proteinase1918C-proteinaseBMP1-3propeptideCProcollagen Io95450958O95450-1ER signal,Proteinase2120N-proteinaseLpNPIpropeptideNLysylo60568714OneER signalLH32322hydroxylase 3version

[016...

example 3

Detection of Human Collagen in Transgenic Tobacco Plants

[0183]Total soluble proteins were extracted from tobacco transformants 2, 3 and 4 by grinding 500 mg of leaves in 0.5 ml 50 mM Tris-HCl pH=7.5 with a “Complete” protease inhibitor cocktail (product #1836145 from Roche Diagnostics GmbH, 1 tablet per 50 ml buffer). The crude extract was mixed with 250 μl 4× Sample application buffer containing 10% beta-mercapto-ethanol and 8% SDS, the samples were boiled for 7 minutes and centrifuged for 8 minutes in 13000 rpm. 20 μl of the supernatant were loaded in a 10% polyacrylamide gel and tested with anti-Collagen I (denatured) antibody ((#AB745 from Chemicon Inc.) in a standard Western blot procedure (FIG. 4). W.T. is a wild type tobacco. Positive collagen bands are visible in plants that are PCR positive for collagen typeI alpha 1 or alpha 2 or both. Positive control band of 500 ng collagen type I from human placenta (#CC050 from Chemicon Inc.) represents about 0.3% of the total soluble ...

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Abstract

A method of producing collagen in a plant and plants producing collagen are provided. The method is effected by expressing in the plant at least one type of a collagen alpha chain in a manner enabling accumulation of the collagen alpha chain in a subcellular compartment devoid of endogenous P4H activity, thereby producing the collagen in the plant.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 11 / 730,071 filed on Mar. 29, 2007, which is a continuation-in-part of PCT Patent Application No. PCT / IL2005 / 001045 filed on Sep. 28, 2005, which claims the benefit of priority of U.S. Provisional Patent Application No. 60 / 613,719 filed on Sep. 29, 2004. The contents of all of the above applications are incorporated by reference as if fully set forth herein.FIELD AND BACKGROUND OF THE INVENTION[0002]The present invention relates to collagen producing plants and methods of generating and using same. More particularly, the present invention relates to a novel approach for generating plants capable of producing high levels of hydroxylated collagen chains which are capable of forming native triple helix type I collagen fibers.[0003]Collagens are the main structural proteins responsible for the structural integrity of vertebrates and many other multicellular organisms. Type I collagen represen...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/82A01H1/02A01H5/00
CPCC12N15/8257C12P21/02C07K14/78C12N9/0071C12Y114/11002C12Y114/11004
Inventor SHOSEYOV, ODEDSTEIN, HANAN
Owner COLLPLANT LTD
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