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.
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1. Substantially pure gelonin toxin having the amino acid sequence:
PAR
or a fragment or derivative thereof, said fragment or derivative having an activity which inhibits cellular protein synthesis but does not bind to a cell surface receptor. 2. A dna sequence of the formula:
PAR
or a fragment or derivative thereof, said fragment or derivative coding for gelonin or for a polypeptide having an activity which inhibits cellular protein synthesis but does not bind to a cell surface receptor. 3. The toxin of
4. The dna sequence of
5. The gelonin toxin of
6. The gelonin toxin of
8. The gelonin toxin of
9. The gelonin toxin of
10. The gelonin toxin of
11. The gelonin toxin of
12. The gelonin toxin of
15. The gelonin toxin of
16. The gelonin toxin of
17. The gelonin toxin of
18. The gelonin toxin of
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This is a and 6D , 6D and 6E demonstrate the hydrophobicity plots of gelonin (A), trichosanthin (B), abrin (C), ricin (D), and agglutinin precursor (E).
The term "substantially pure" when applied to the gelonin protein of the present invention means that the polypeptide is essentially free of other plant proteins normally associated with the gelonin in its natural state and exhibiting reproducible electrophoretic or chromatographic response, elution profiles, and toxic activity. The term "substantially pure" is not meant to exclude artificial or synthetic mixtures of the gelonin protein with other compounds.
Gelonin was purified from the seeds of the plant Gelonium multiforum by techniques known to those of skill in the art. The amino acid sequence was determined utilizing a modification of the Edman degradation method.
Samples of gelonin were applied to the reverse phase reaction chamber and subjected to Edman degradation. The N-terminal of gelonin was found to be heterogeneous (1/2 of the molecules of the protein were apparently one amino acid shorter than the others). This heterogeneity made it difficult to sequence much more than 40 cycles. Therefore, in order to determine further amino acids in the sequence, enzymatic cleavage was performed.
Internal sequence of proteins is generally obtained by digesting or cutting up the large protein molecule into smaller pieces with a combination of enzymes and chemical cleavages. When native gelonin was exposed to various protolytic enzyzme digestions, it was found to be incompletely cleaved. This was found to be partly due to a disulfide bond in the N-terminal part of the molecule. Breaking of this bond by reduction and alkylation with iodoacetic acid yielded a fragment that was less soluble than the native material at the pH required for enzymatic digestion. A combination of digestion of native gelonin with trypsin, Lysine aminopeptidase (Lysc), staphylococcal protease (V8), and chymotrypsin yielded peptides mostly from the C-terminal portion of the molecule. This indicated that the N-terminal part of the molecule (from the N-terminal analysis to the Asp-Ala-Pro at residue 70) was not readily accessible by enzyme digestions.
Gelonin was cleaved with cyanogen bromide into 3 large peptides. Protein aliquots (0.2 mg/ml) were dissolved in 70% formic acid. A crystal of cyanogen bromide was added to the solution and the reaction allowed to proceed for at least 18 hours. The solution was then diluted with water and was applied to a small sequencing column. After sample application, a gradient of 1% to 10% n-propanol with 0.1% TFA was used to elute the protein fragments. The elution profile is shown on FIG. 4.
Enzymatic digestion of the whole protein or of CNBr fragments yeilded overlapping peptides. Enzymatic digestions with Lysyl endopeptidase in 0.1% SDS 100 mM Tris pH 8.0, Staphylococcus aureus protease in 0.1% SDS or trypsin in 0.1% Tween 20 were carried out. Gelonin contains one cysteine residue at position 49. Reduction and carboxymethylation yields a protein which recovers better on reverse phase HPLC and is more susceptible to enzymatic digestion. Therefore, most of the enzymatic digestions were carried out in 0.1% SDS or 0.1% Tween.
After the C-terminal 160 residues were aligned by a combination of CNBr digests and enzymatic cleavages, the remaining unknown sequence between residues 40 to 70 was determined by a combination of chemical modification of cysteine with iodoacetic acid and solubization of the alkylated protein with SDS. The RCM alkylated gelonin was then cleaved with excess Lysc enzyme at 37°C for short periods of time (1-5 hr.). The HPLC elution profile is shown on FIG. 5A.
This method yielded a new sequence that had not been seen before. This new sequence showed the existence of an Asn-Gly combination. This combination of amino acids is cleaveable by a chemical method using hydroxylamine.
Hydroxylamine cleavage was carried out by adding 100 ug of gelonin to freshly prepared hydroxylamine (2M) in 0.2 M Tris (pH 9.0) with 2M NaCl, 1 mm EDTA and 10% ethanol. After incubation for 7 hours at room temperature, the entire reaction mixture was applied to a sequencing column. The column was then washed with 1% TFA in water and either eluted with an acetonitrile gradient or was sequenced directly as a mixture. This chemical cleavage produced a large hydrophobic peptide that contained about a 200 amino acid sequence which connected with the Asp-Ala-Pro at residue 70. The elution profile is shown on FIG. 5C.
The remaining, short section of overlapping sequence from between residues 40 to 50 was determined by digesting gelonin without alkylation by Lysc in SDS. This digested away most of the C-terminal part of the material. Then this mixture was digested again by chymotrypsin. The products of this digestion were then separated by HPLC. Sequence analysis of a large peptide revealed a sequence (SerThrLys) starting about 5 amino acids in from the N terminal end of the molecule. This was useful in that it removed the heterogeneous part of the molecule and allowed for a longer sequence run.
Gelonin protein comprises 258 amino acids, the sequence of which is demonstrated on FIG. 1. The amino acid sequence of gelonin was compared to other known sequences available in sequence data banks (Genbank, PIR, EMBL) to determine whether gelonin has any areas of homology with other proteins. Comparison of the gelonin amino acid sequence with other proteins having known amino acid sequences demonstrated that the gelonin sequence is unique. Homology of certain portions of the gelonin sequence to portions of other proteins was detected. For instance, gelonin demonstrates a 36.0% homology with alphatrichosanthin from Trichosanthin kirilowi, 33.8% homology with Abrin A chain from Indian Liquorice, 35.2% homology with agglutinin precursor from Castor bean, 33.7% homology with Ricin D, A chain from Castor bean and 27.3% homology with antiviral protein (MAP) from Mirabills jalapa. A summary of the degree of homology to these and other proteins is shown on FIG. 3.
Hydrophobicity plots shown on FIGS. 6A-6E demonstrate a similarity to hydrophobic regions of trichosanthin, Ricin and to other ribosomal inhibiting proteins.
A plot of the hydropathy of the gelonin structure shows a hydrophobic region in residues 35-80 and 150-180. These are areas in which substantial folding of the molecule probably occurs. This similar hydrophobic pattern is also observed for other toxins (see FIGS. 6A-6E) and may suggest that the active enzymatic center may be contained within these folded regions. Therefore, the active enzymatic site may not be found in a linear region of the molecule and these structures may need to be adequately folded to attain the proper enzymatic center.
Utilizing the cDNA of gelonin, recombinant gelonin can be produced. Mutations can be specifically introduced into the molecule in order to provide recombinant gelonin lacking carbohydrate groups which can misdirect gelonin-antibody conjugates. Recombinant gelonin molecules can be produced by site directed mutagenesis to have greater toxic activity than the native molecule, to be more effectively internalized once bound to the cell surface by a carrier such as a monoclonal antibody or a targeting ligand such as IL-2, EGF, IFN, etc., to resist lysosomal degradation and thus be more stable and longer acting as a toxic moiety.
Recombinant gelonin molecules can also be engineered as fusion products to contain other functional modalities to kill cells such as an enzymatic activity, TNF, IFN activity, a second toxic activity, such as diptheria toxin action (wherein said second activity was through a different biological pathway than gelonin), thus creating a "supertoxin" or a toxin with multifunctional actions.
Fusion proteins can be engineered with gelonin to carry drugs such as chemotherapeutic agents or isotopes for radioimaging or radiotherapy. Gelonin peptides may have application as abortofacient agents, immuno suppressive agents, anticancer agents and as antiviral agents (such as an anti-HIV agent).
The following examples provide a detailed description of the preparation, characterization, and amino acid sequence of gelonin. The experimental methods utilized are described in detail in the examples below. These examples are not intended to limit the invention in any manner.
PAC Purification and Characterization of GeloninGelonin 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 mM sodium phosphate (pH 7.4). The homogenate was left overnight at 4°C with continuous stirring, cooled on ice and centrifuged at 35,000 times g for 20 minutes at 0°C The supernatant was removed, dialyzed against 5 mM sodium phosphate (pH 6.5) and concentrated using a pm10 filter. The sample was layered on a CM-52 ion-exchange column (20×1.5 cm) equilibrated with 5 mM sodium phosphate (pH 6.5). Material which bound to the ion exchange resin was eluted with 400 ml of 0 to 0.3 M linear NaCl gradient at a rate of 25 ml hour at 4°C Five ml fractions were collected. The fractions were monitored at 280 nm in a spectrophotometer. The gelonin eluted in about fractions 55-70 and was the last major elution peak. These fractions were pooled, dialyzed against 0.1 M NaCl in 0.1 M Na2 HPO4 buffer (pH 7.4). The sample was then applied to a Cibacron blue sepharose column (24×2 cm) previously equilibrated with 0.1 M Na2 HPO4 /0.1 M NaCl buffer. The column was washed with 3 column volumes of buffer and eluted with a 400 ml linear salt gradient (from 0.1 M NaCl to 2 M NaCl). Elution of the bound material was monitored by Lowry assay of the column fractions. The fractions containing the single protein peak were pooled and dialyzed overnight at 4°C against PBS. Gelonin toxin was purified to greater than 97% purity as estimated from silver stained PAGE. The purity and the molecular weight of each preparation was checked on high pressure liquid chromatography using a TSK 3000 gel permeation column with 50 mM sodium phosphate buffer, pH 7.4 and 15% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-page). Gelonin migrated as a single band with an approximate molecular weight of 29-30,000 daltons.
PAC Assay of Gelonin ActivityThe 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(CH3 CO2)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 [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°C 14 C-leucine incorporation was monitored in an aliquot of the mixture by precipitating synthesized protein on glass fiber filters, washing in 10% TCA and acetone, and monitoring the radioactivity in a Beta-counter using Aquasol scintillation fluid. Utilizing this assay, purified gelonin had a specific activity of 4×109 U/mg protein. A unit of gelonin activity is the amount of gelonin protein which causes 50% inhibition of incorporation of [14 C] leucine into protein in the cell free assay.
PAC Determination of Gelonin Amino Acid SequenceThe 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 H2 O at 65°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 (cleaves only after lysine residue). The protein was found resistant to as much as 5% (w/w) of the enzyme. The resistance of gelonin to the proteolytic enzyme trypsin is not due to a lack of trypsin cleavage sites, since gelonin contains 21 lysine and 12 arginine residues. These results indicate that gelonin is perhaps a rigidly packed molecule which makes it inaccessible to proteolytic enzymes.
Since gelonin was found resistant to cleavage by proteolytic enzymes, chemical cleavage of the protein was examined.
PAC CNBr Cleavage of GeloninGelonin prepared as in Example 1 was dissolved in 70% formic acid. A crystal of cyanogen bromide was added to the solution. After at least 18 hours the solution was applied to either a small column (0.15 cm×5 cm) reverse phase (J. T. Baker, 15 cm C-1B bonded phase Cat II 7191-02) or analytical (4.6×100 mm) reverse phase column. A gradient elution of 1 to 70% n propanol with 1% TFA in water produced 5 peaks as shown on FIG. 6. Each of the peaks were sequenced and also used for further digestion by enzymes to piece together the entire sequence. Peak 1 was sequenced directly and gave a sequence starting with a Phe (F) that ran for 38 residues and ending with a Glu (E). This sequence was confirmed by mass spectroscopy and Lysc digestions of this isolated peptide. Peak 2 was sequenced directly and gave a sequence starting with a Val (V) that ran for 47 cy and was not interruptible after the Ala at cy 47. Peak 3 was sequenced and gave the same sequence as peak 2. SDS gels of peaks 2 and 3 as well as Lysc digestion of peaks 2 and 3 showed that peak 3 contained the C-terminal CNBr peptide as well. Subsequent trypsin digestion of gelonin produced a peptide that connected these two CNBr peptide sequences. This trypsin peptide when sequenced gave the sequence TSGANGMFSEAVELER. Peak 4 and 5 both gave the N-terminal sequence GLDT . . . . This was used for some digestion by Lysc, 1/8, to give peptides from its C-terminal end.
PAC Enzymatic Digestion of CNBr Cleaved GeloninSamples of whole protein or CNBr fragments were digested with Lysyl endopeptidase (Wako Chemical Dallas, Tex.) in 0.1% SDS 100 mM Tris pH 8.0 or Staphylococcus aureus protease (Pierce) in 0.1% SDS or Trypsin (Sigma) in 0.1% Tween 20. Digestion mixtures were separated by HPLC and collected peptides were sequenced on the prototype sequence using gas-phase Edman sequencing methods.
PAC Amino Acid Sequence of GeloninA total of 258 amino acid residue sequences were obtained following analysis of the CNBr fragments obtained in Example 3. FIG. 1 shows the amino acid sequence of gelonin. Gelonin contains a total of approximately 258 amino acid residues. The DNA sequence was deduced from this amino acid sequence. The degenerate DNA sequence is shown on FIG. 2. Those skilled in the art will recognize that fragments and derivatives of either the gelonin amino acid sequence or the DNA sequence coding for gelonin may inhibit cellular protein synthesis but not bind to a cell surface receptor.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth below.
Rosenblum, Michael, Kohr, William J., Aggarwal, Bharat
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