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Protein sequence of the plant toxin gelonin

Inactive Publication Date: 2001-12-04
RES DEVMENT FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A major challenge for the design of a drug for treatment of any disease is specificity and efficacy.
The availability of a toxin molecule which is not cytotoxic to a variety of cells when administered alone has been limited.
However, the availability of these single chain toxins in substantially pure form is limited due to the fact that they must be purified from plant sources in which they occur in relatively low amounts and the reproducibility of the concentration of the toxin in the plants is dependent upon plant growth conditions and plant harvest conditions.
However, the availability of a reproducible, readily accessible supply of gelonin from natural sources is limited.
In addition, the purification of gelonin from plant sources is difficult and the yield is very low.
However, chemical modification of gelonin and cellular targeting moieties can reduce targeting efficiency and cytotoxic potential of gelonin itself.
Furthermore, natural sources of gelonin are subject to variability in harvesting and plant growth which can affect gelonin cytotoxic activity.

Method used

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  • Protein sequence of the plant toxin gelonin
  • Protein sequence of the plant toxin gelonin
  • Protein sequence of the plant toxin gelonin

Examples

Experimental program
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Effect test

example 1

Purification and Characterization of Gelonin

Gelonin was isolated from the seeds of the plant Gelonium multiforum essentially according to the procedure as described (Stirpe, et al. (1980) J. Biol. Chem 255 6947-6953). Briefly, gelonin was extracted from the seeds by homogenization in buffered saline solution (pH 7.4). The supernatant was concentrated after dialysis against 5 mM sodium phosphate (pH 6.5) and the gelonin further purified by ion exchange chromatography as described below. The purity of the gelonin toxin was assessed by high pressure liquid chromatography (HPLC) and sodium dodecylsulphate-polyacylamide gel electrophoreseis (SDS-Page). Gelonin toxin migrated as a single band with an approximate molecular weight of 29-30,000 daltons.

Gelonin toxin activity was measured as described in Example 2 by protein synthesis inhibition in a cell-free system.

Seeds of Gelonium multiforum were shelled and the nuts ground in a homogenizer with eight volumes of 0.14 M NaCl containing 5 m...

example 2

Assay of Gelonin Activity

The gelonin activity was monitored in a cell-free protein synthesis inhibition assay. The cell-free protein synthesis inhibition assay was performed by sequentially adding to 50 ul rabbit reticulocyte lysate, thawed immediately before use, mixing after each addition, the following components: 0.5 ml of 0.2 M Tris HCl (pH 7.8), 8.9 ml of ethylene glycol, and 0.25 ml of 1 M HCl).

Twenty microliters of a salt-amino acid-energy mixture (SAEM) consisting of: 0.375 M KCl, 10 mM Mg(CH.sub.3 CO.sub.2).sub.2, 15 mM glucose, 0.25-10 mM amino acids (excluding leucine), 5 mM ATP, 1 mM GTP, 50 mM Tris-HCl (pH 7.6), 10 ul Creatinine phosphate-creatinine phosphokinase, 8 ul [.sup.14 C] leucine (Amersham, 348 mCi / mmol), and adding 1.5 ul of solutions containing varying concentrations of the gelonin mixture. The mixture was incubated for 60 minutes at 30.degree. C. .sup.14 C-leucine incorporation was monitored in an aliquot of the mixture by precipitating synthesized protein ...

example 3

Determination of Gelonin Amino Acid Sequence

The gelonin amino acid sequence was determined by the Edman degradation method using an automated amino acid sequencer as described in European Patent Application No. EP-257735. Large peptides and unfragmented protein were applied to the reverse phase portion of the sequence reaction chamber. Unwanted buffer components were washed off with excess water. The protein or peptide sample was then sequenced by Edman chemistry and the extracted ATZ amino acid derivatives were converted to the PTH form by 25% TFA in H.sub.2 O at 65.degree. C. PTH samples were identified by reverse phase analytical separation on a Np 1090 column.

In order to obtain further amino acid sequence, the protein was digested with various proteolytic and chemical agents and then the peptides were purified by high performances liquid chromatography. Gelonin was found quite resistant to the exposure of trypsin (cleaves after arginine and lysine residues) and acetyl trypsin (c...

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Abstract

This invention relates to substantially purified gelonin, toxic fragments thereof, the DNA sequences encoding gelonin and use of the DNA for producing, by recombinant technology, gelonin, toxic fragments thereof and fusion proteins. More specifically, the invention relates to the primary amino acid sequence of gelonin, and of the DNA encoding said gelonin and the production of synthetic gelonin and toxic fragments thereof.

Description

TECHNICAL FIELDThis invention relates to substantially purified gelonin, toxic fragments thereof, the DNA sequences encoding gelonin and use of the DNA for producing, by recombinant technology, gelonin, toxic fragments thereof and fusion proteins. More specifically, the invention relates to the primary amino acid sequence of gelonin, and of the DNA encoding said gelonin and the production of synthetic gelonin and toxic fragments thereof.BACKGROUND ARTA major challenge for the design of a drug for treatment of any disease is specificity and efficacy. Various drugs available for the treatment of cancer suffer from problems of this nature. The concept of targeting toxic drugs selectively to certain tumors has been a subject of intense research in the last few years (Thorpe (1985) Biol Clin Applications 84:475-512; Moller ed. (1982) Immun. Rev. 62:1-215). Recently both monoclonal and polyclonal antibodies, lectins, lymphokines and hormones which recognize specific determinants on the su...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/62A61K38/00A61K36/47A61K38/16A61K39/395A61P35/00C07K1/00C07K14/00C12N15/09
CPCC07K14/415C07K2319/55C12N15/62A61P31/00A61P35/00A61P37/06C07K14/00
Inventor ROSENBLUM, MICHAELKOHR, WILLIAM J.AGGARWAL, BHARAT
Owner RES DEVMENT FOUND
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