WO1991000346A1

WO1991000346A1 - Polynucleotide phosphorylase immobilized on tris(hydroxymethyl)methylacrylamide polymer beads

Info

Publication number: WO1991000346A1
Application number: PCT/US1990/003670
Authority: WO
Inventors: Jose Ricardo Matos; John Richard Moran
Original assignee: E.I. Du Pont De Nemours And Company
Priority date: 1989-07-03
Filing date: 1990-07-03
Publication date: 1991-01-10
Also published as: AU5943690A

Abstract

Polynucleotide phosphorylase immobilized by covalent bonding to particles of a water-insoluble polymer of tris(hydroxymethyl)methylacrylamide is used to catalyze synthesis of polyribonucleotides from nucleotide diphosphates or production of nucleotide diphosphates by phosphorolysis of polyribonucleotides.

Description

TITLE POLYNUCLEOTIDE PHOSPHORYLASE IMMOBILIZED ON TRIS(HYDROXYMETHYL)METHYLACRYLAMIDE POLYMER BEADS FIELD OF THE INVENTION

This invention relates to immobilized polynucleotide phosphorylase (PNPase) for the preparation of polyribonucleotides.

BACKGROUND OF THE INVENTION

Polynucleotide phosphorylase is an enzyme which catalyzes the synthesis of single strand polyribonucleotide (also called polyribonucleoside monophosphate, poly NMP or NMP_n) from ribonucleotide diphosphates (NDP's) in the presence of magnesium ions:

NDP + (NMP)_n_i < > (NMP)_n + Pi

where n is an integer and P-j represents phosphate ion. Each NDP is added sequentially to the free 3'-hydroxyl terminus of previously formed polyribonucleotide (NMP)_n_ι. Frequently, a primer strand of (NMP)_n_ι is used at the beginning of the reaction. Reaction equilibrium can be shifted in the direction of breakdown of polyribonucleotide by increasing the phosphate ion concentration. Thus, PNPase can be used to catalyze the phosphorolysis of polyribonucleotides to produce ribonucleoside diphosphates as well as to catalyze the production of polyribonucleotides from ribonucleoside diphosphates.

The poly NMP can be a naturally occurring single strand ribonucleic acid (ssRNA) or a homopolymer or copolymer ssRNA such as polyadenylic acid (poly-A), polyinosinic acid (poly I), polycytidylic acid (poly C) or polycytidylic/uridylic acid (poly C,U). Poly I and poly C or poly C,U can be annealed to form double stranded ribonucleic acid (dsRNA) useful for induction of interferon and in the treatment of viral infections and cancer, as disclosed, for example in U.S. Patent 4,130,641 and European Published Applications 113,162 and 213,921. The synthesis and phosphorolysis reactions are normally carried out with the PNPase in solution as the free enzyme. However, as described in Boyer, Editor, The Enzymes. 3rd Edition, volume XV, part 8, (1982), page 546, PNPase has been immobilized on various insoluble materials such as nitrocellulose filters, cellulose beads, mercerized cellulose, Sepharose® 4B polysaccharide, hydrazide agarose, diazotized p-aminobenzenesulfonylethyl (ABSE) agarose, ABSE Sephadex® G-200 and ABSE cellulose. Boyer states that the immobilized PNPase has advantages over the soluble enzyme; yield of polymerization is improved with some supports under pH conditions which favor polymerization over phosphorolysis, separation of reaction products from enzyme is simpl fied, and the enzyme can be recycled multiple times. Thang et al., Biochemical and Biophysical Research Communications, Vol. 31, pp. 1-8 (1968) discloses PNPase immobilized on a nitrocellulose filter. Hoffman et al., Biochemical and Biophysical Research Communications, Vol. 41, No. 3, pp. 710-714 (1970) discloses use of PNPase bound to CNBr activated cellulose for production of homopolynucleotides. Smith et al., FEBS Letters, Vol. 30, No. 2, pp. 246-248 (1973) discloses use of PNPase bound to mercerized cellulose or Sepharose® or a inoalkylsilane glass beads for preparation of single strand polyribonucleotides. Soreq et al., Journal of Biological Chemistry, Vol. 252, pp. 6885-6888 (1977), discloses PNPase immobilized onto CNBr activated Sepharose® for phosphorolysis of polynucleotides. Bachner et al., Biochemical and Biophysical Research Communications, Vol. 63, No. 2, pp. 476-483 (1975) mentions that polynucleotides were synthesized with PNPase immobilized on agarose. Yamauchi et al., Journal of Fermentation Technology, Vol. 64, No. 6, pp. 517-522 (1986) and Yamauchi et al . , Journal of Fermentation Technology, Vol. 64, No. 5, pp. 455-457 (1985) disclose PNPase immobilized on a inopropyl porous glass by various covalent bonding methods, and states that the product obtained by means of glutaraldehyde bonding is suitable for practical production of polynucleotides such as poly I and poly C. Yamauchi also refers to prior methods of immobilizing PNPase on CNBr activated cellulose and on silica gel, but states that these methods were not entirely satisfactory. Brentnall et al., Tetrahedron Letters, No. 25, pp. 2595-2596 (1982) discloses polymerization of 8-chloroadenosine diphosphate using PNPase immobilized on cellulose. Vang et al., Biochemical and Biophysical Research Communications, Vol. 90, No. 2, pp. 606-614 (1979) discloses immobilization of PNPase on CNBr activated Sepharose® and states that the bound enzyme retains 70% of its activity in polymerization of NDP's. Cashion et al . , Biotechnology and Bioengineering, Vol. XXIV, pp. 493-423 (1982), Cashion et al., Nucleic Acids Research, Symposium Series No. 7, pp. 173-189 (1980), and Cashion et al . U.S. Patent 4,379,843 describe the immobilization of PNPase and other enzymes on tritylated agarose or Sepharose® beads by non-covalent bonding. Ansberga et al . , U.S.S.R. published application 810,717 discloses PNPase immobilized in gamma aluminum hydroxide for synthesis of polynucleotides. Kratasyuk et al., Izv. Sib. Otd. Akad. SSSR. Ser. Biol. Nauk., Vol. 2, pp. 93-99 (1978) discloses PNPase immobilized on a macroporous matrix for synthesis and phosphorolysis of polynucleotides. Kumarev et al., Bioorg. Khim., Vol. 2, pp. 700-707 (1976) discloses immobilization of PNPase on a propionaldehyde- containing silochrome support. Broom, A.D., in an unpublished personal communication, describes polynucleotide synthesis experiments using PNPase immobilized on a number of different supports, Including derivatized cellulose, polyacrylamide, and agarose; the commercially available support which gave best results was Affi-Gel®10 (Bio-Rad), a succinimide derivative of agarose, but even better results were obtained with a benzyl derivative of Sepharose®4B, which bound PNPase hydrophobically rather than covalently.

Epoxy-activated particles, i.e., particles of polymers that contain oxirane groups as active protein-binding groups, have been used for immobilization of various enzymes. Kraemer et al. US patents 4,070,348, 4,190,713, 4,208,309, 4,247,643 and 4,511,694 disclose particles of this type and thei use to bind enzymes. The Kraemer et al . particles are composed of copoly ers of (A) an ethylenically unsaturated monomer having a glycidyl group, (B) a comonomer having at least two radical polymerizable double bonds, and (C) a radical- polymerizable water soluble comonomer. Matthews U.S. Patents 3,844,892 and 3,932,557 disclose immobilization of enzymes on epoxy- containing copolymers prepared by reacting a monomer containing a 1,2-epoxy group and terminal unsaturation with an olefin monomer such as acrylonitrile, ethacrylonitrile, methyl acrylate or methyl methacrylate.

Bigwood U.S. Patents 4,582,860 and 4,612,288 disclose immobilization of enzymes on epoxy- activated polymer beads made by copolymerizing an oxirane-bearing onovinyl monomer, such as glycidyl acrylate or methacrylate or allyl glycidyl ether, with a trivinyl monomer such as tri ethylolpropane trimethacrylate. Tris(hydroxymethyl)methylacrylamide polymers have found extensive use in the area of chromatography. These polymers, which are marketed by IBF Biotechnics (Savage, MD) under the trademark TRISACRYL® are homopolymers of tris(hydroxymethyl)methylacrylamide (alternative name: N-acry1o 1-2-amino-2-hydroxymeth 1- 1,3-propanediol) or copolymers of tris(hydroxymethyl)methylacrylamide with one or more secondary monomers such as acrylic acid, amino and quaternary ammonium aery1amide derivatives and sulfonic acid acrylamide derivatives. Examples of specific secondary monomers include diethylaminoethylacrylamide (alternative name: N- acryloyl-l-anrino-2-diethylaminoethane) and quaternary ammonium salts thereof with ethyl chloride and diethylaminoethyl chloride, and N- acr 1oy1-2-amino-2-meth 1-1- propanesulfonic acid. IBF Biotechnics teaches, in its product literature, the use of these supports for desalting of proteins and gel filtration, 1on exchange, affinity and size exclusion chromatographies. TRISACRYL® GF 2000 homopolymer of tris(hydroxymethyl)methylacrylamide has found use in enzyme immobilization. It has been activated with carbonyldiimidazole for subsequent use in reversible enzyme immobilization (Editor K. Mosbach, Methods in Enzymology 1987, Vol. 135, part B, pp. 33 and 109; M. T. W. Hearn, J. of Chromatography 1986, Vol. 376, p. 245). The resulting immobilized protein was used as an affinity support. The homopolymer has also been activated with 2-fluoro-l-methylpyridinium tosylate and the resulting polymer sold under the trade name AvidGel®T by Bioprobe International (Tustin, CA) . AvidGel®T is marketed as an affinity chromatography support for the irreversible binding of ligands. Although immobilization of PNPase provides several advantages over the use of free enzyme for synthesis of polyribonucleotides, immobilization on certain supports can present several problems, including loss of enzyme activity upon immobilization and leaching of enzyme from the support under the polymerization conditions. For example, Schnapp et al., Biochemical and Biophysical Research Communications, Vol. 70, No. 1 (1976) refers to the problem of leaching of enzyme from CNBr activated Sepharose®, and suggests instead the use of amine, hydrazine or hydrazide carrying supports to alleviate the problem.

SUMMARY OF THE INVENTION We have discovered that PNPase can be immobilized on particles of the water insoluble tris(hydroxymethyl)methylaery1amide polymers to provide immobilized catalyst of high specific activity which resists leaching and retains enzyme activity after repeated use. Rigid non-porous or macroporous particles of tris(hydroxymethyl)methylacrylamide allow use of the immobilized PNPase in various reactor designs, e.g., batch type reactor, fluidized bed reactor, packed bed reactor or continuous flow reactor. The immobilized PNPase exhibits a specific activity in the range of 0.1 - 1.5 IU/g of wet support. The specific activity is defined as units of PNPase activity. A unit (IU) of activity is that activity which will produce 1 /imole of inorganic phosphate in 1 minute at 25°C under the assay conditions described below. PNPase is leached from the tris(hydroxymethyl)methylacrylamide support at a rate < about 5%, generally <1% in continuous use over 5 cycles under standard polyribonucleotide synthesis conditions (pH 9, 25°C). The immobilized enzyme retains at least 90% of its original activity after being used 5 times for polyribonucleotide synthesis. The low leaching rate was unexpected, because PNPase is a tri er composed of three polypeptide subunits. It is unknown whether all three subunits are covalently attached to the tris(hydroxymethyl)methylacrylamide activated support particles.

DESCRIPTION OF THE FIGURES Fig. 1 is a plot showing the release of N- methyl-2-pyrrolidone from the support during the immobilization of PNPase on AvidGel®T particles as described in Example 1 reaction. Fig. 2 is a plot showing the consumption of inosine diphosphate and formation of the polyribonucleotide poly-I as described in Example 2. DETAILED DESCRIPTION Tris(hydroxymethyl)methylacrylamide polymer particles suitable for use in this invention are described by IBF Biotechnics in their TRISACRYL® product literature. They are homopolymers of tris(hydroxymethyl)methylacrylamide or copolymers of tris(hydroxymethyl)methylacrylamide and acrylic acid or an amino or a quaternary ammonium acrylamide derivative or a sulfonic acid acrylamide derivative. Examples of specific secondary monomers are those mentioned above. Tris(hydroxymethyl)methylacrylamide polymer particles can be activated by reaction with bisepoxiranes, epichlorohydrin, glutaraldehyde, N- ethoxycarbony1-2-ethoxy-l,2-dihydroquinoline, carboxyl anhydride, divinyl sulfone, tosyl chloride, acyl chloride or 2-fluoro- l-methylpyridinium tosylate. The resulting activated particles are accessible to reactive nucleophilic functional groups of a protein, e.g., an amine, sulfhydryl, alcohol, acid, amide, i idazole, or aromatic carbon group. Especially suitable for the immobilization of PNPase is the tris(hydroxymethyl)methylacrylamide homopolymer that has been activated with 2-fluoro- l-methylpyridinium tosylate; this activated polymer is commercially available as AvidGel®T from BloProbe International (Tustin, CA).

Average particle size of the support is not critical, but will generally be in the range of about 1 to 1,000 μm. The particles can be substantially non-porous or can contain pores (channels and cavities) with average diameter in the range of about 0.01 to 1 μm. Particles In the average size range of about 10 to 500 μm containing pores with average diameter in the range of about 0.02 to 0.5 μm are preferred for preparation of polyribonucleotides with chain length in the range of about 200 to 1,000 nucleotides. This is the preferred ssRNA chain length range for producing dsRNA's with higher pharmacological activity and lower toxicity. Most preferred at present are supports with average bead size in the range of about 50 to 150 μm and average pore size in the range of about 0.02 to 0.2 μm.

Immobilization of the PNPase on the activated tris(hydroxymethyl)methylacrylamide beads is accomplished by mixing the beads with an aqueous buffered solution of PNPase, adjusting to the desired pH, and letting the mixture stand for a few (e.g., 10-24) hours, preferably with mild agitation. The immobilization can be carried out at pH in the range 7.5 to 9.5, but pH in the range of 8 to 9 is preferred for obtaining optimum specific activity and yield. If desired, the beads can be swollen by soaking in buffer before mixing with PNPase.

In the examples below, the following General Methods were used.

Enzyme Assays

The enzymatic activity of both the soluble and the immobilized form of PNPase were measured by incubating the enzyme (ca. 30 mg of crude protein/ L for the soluble enzyme; 100 mg of wet solid for the immobilized enzyme) in a solution containing tris buffer (0.1M, pH 9.0), inosine diphosphate (IDP, 20 mM) , MgCl₂ (5 mM) at 25°C for 1 hour. The inorganic phosphate produced in the reaction is measured by the method of Bencini et al., Anal. Biochem., Vol. 132, pp. 254-258 (1983). A unit of activity (IU) of PNPase is defined as that activity which will produce 1 /nmol of inorganic phosphate in 1 minute under the assay conditions.

HPLC Analysis

HPLC analyses were performed on a Hewlett- Packard 1090 M HPLC equipped with a UV diode array detector and a Du Pont GF-450 size exclusion column. The elutant is Na2HP04 (0.2M, pH 7.0) at a flow rate of 1.0 mL/min. The polynucleotides and the nucleoside diphosphates are separated under these conditions.

EXAMPLES

Example 1: PNPase Immobilized on AvidGel®T Particles

AvidGel®T particles (10.0 g dry weight) were suspended in aHCθ3 buffer (50 mL, 50 mM, pH 8.5) and tumbled for 15 min; the supernatant was then filtered off. A buffered solution of PNPase (8.3 mL, 700 mg of crude protein, 250 IU/mL) was mixed with NaHC0₃ buffer (30 mL, 0.05 M pH 8.5) and

AvidGel®T particles above (10.0 g wet weight). The pH was adjusted to 8.5 with 0.1 N NaOH, and the mixture was tumbled at room temperature for 20 h. The immobilization reaction was monitored by UV (297 nm) absorbance measurement of the supernatant as the N-methyl-2-pyrrolidone was released from the support (see figure 1). The pH was kept constant at 8.5 with 0.1 N NaOH. The supported catalyst was filtered and washed (3 X 30 mL) with Tris ethanola ine buffer (0.1 M:0.1 M, pH 8.0). The supported catalyst was suspended in the Tris- ethanolamine buffer (30 mL) and tumbled at room temperature overnight; the quench reaction can also be monitored by UV (297 n ) . The filter/wash procedure was repeated using Tris buffer (7 X 30 L, 0.1 M pH 9.0) and the final support was suspended in this Tris buffer (50 L). Immobilization yields: 1) protein bound >90%; 2) activity bound 90 %; 3) specific activity = 1.5 IU/g of wet support.

Example 2: Multiple Preparation of Polynucleotides with Immobilized PNPase

PNPase immobilized on AvidGel®T particles (15 g wet weight) was suspended in Tris buffer (90 mL, 0.1 M pH 9.0) containing inosine diphosphate (IDP, 60 M) and MgCl£ (5 mM) . The reaction was tumbled slowly at 24°C for 20 h, and could be monitored by SEC HPLC, above, by taking aliquots at regular intervals (see figure 2). The suspension was filtered (0.45 μ polyester filter) and the support washed with Tris buffer (25 L, 0.1 M pH 9.0). The recovered immobilized enzyme retained >90% of its original activity. The above filtrates were combined and ultrafiltered with a Millipore MINITAN® ultrafiltration apparatus equipped with a polysulfone membrane (10,000 Mw cutoff) and peristaltic pump. The retentate was recycled until its total volume was <20 mL. The retentate was diluted with EDTA (40 mL, 0.3 M pH 7.0) and then concentrated to <20 mL total volume. This ultrafiltration procedure was repeated once more with this EDTA solution (40 mL) and then sequentially with EDTA (2 X 40 mL, 0.1 M pH 7.0) and distilled/deionized water (2 X 40 mL) . The final retentate was removed from the apparatus and brought to 100 mL total volume with water. Analysis by HPLC indicated the poly inosinic add had been quantitatively retained in the retentate (50-55% overall yield) while the nucleoside diphosphate and other low molecular weight compounds were contained in the filtrate. The aqueous solution of polynucleotide is suitable for lyophilization, precipitation, spray drying or subsequent reaction.

Other homopolymer and copolymer polyribonucleotides can be produced by the method of this example by substituting the appropriate ribonucleoside diphosphate for the IDP. For example, poly C1 U can be made by substituting cytidine diphosphate and uridine diphosphate in the mole ratio 12:1.

Claims

1. Polynucleotide phosphorylase immobilized on particles of a solid or water insoluble polymer of tris(hydroxymethyl)methylacrylamide.

2. Composition of claim 1 wherein the polymer is a homopolymer of tris(hydroxymethyl)-methylacrylamide or a copolymer of tris(hydroxymethyl)methylacrylamide and an amino or quaternary ammonium acrylamide derivative or a sulfonic acid acrylamide derivative or acrylic acid.

3. Composition of claim 2 wherein the polymer particles are about 1 to 1,000 μ in average diameter and contain pores of average diameter in the range of about 0.01 to 1 μm.

4. Composition of claim 3 wherein the particles are about 10 to 500 μm in average diameter and contain pores of average diameter in the range of about 0.02 to 0.5 μm.

5. Composition of claim 4 wherein the polymer is a homopolymer of tris(hydroxymethyl)methylacrylamide or a copolymer of tris(hydroxymethyl)methylacrylamide and N- acryloyl-l-amino-2 diethylaminoethane, or N- acryloyl-2-amino-2-methyl-l-propanesulfonic acid, or acrylic acid.

6. Composition of claim 5 wherein the particles are about 100-300 μm in diameter and contain pores about 0.02 to 0.2 μm in diameter.

7. A method of immobilizing polynucleotide phosphorylase which comprises reacting it in aqueous solution at a pH in the range of 8 to 9 with particles of a solid water-insoluble tris(hydroxymethyl)methylacrylamide polymer.

8. In the method of synthesizing polyribonucleotides from nucleoside diphosphates in the presence of immobilized polynucleotide phosphorylase, the improvement which comprises using polyribonucleotide phosphorylase immobilized by covalent bonding to particles of a solid water- insoluble polymer tris(hydroxymethyl)methyl- acrylamide.

9. Improvement of claim 8 wherein the polymer is a homopolymer of tris(hydroxymethyl)methylacrylamide or a copolymer of tris(hydroxymethyl)methylacrylamide and N- acryloyl-l-amino-2-diethylaminoethane, or N- acryloyl-2-amino-2-methyl-1-propanesulfonic acid, or acrylic acid.

10. Improvement of claim 9 wherein the polymer particles are about 1 to 1,000 μm in average diameter and contain pores of average diameter in the range of about 0.01 to 1 μm.

11. Improvement of claim 10 wherein the particles are about 10 to 500 μm in average diameter and contain pores of average diameter in the range of about 0.02 to 0.5 μm.

12. Improvement of claim 11 wherein the polymer is a homopolymer of tr1s(hydroxymethyl)methylacrylamide or a copolymer of tris(hydroxymethyl) ethylacrylamide and N- acryloyl-l-amino-2 diethylaminoethane, or N- acryloyl-2-amino-2-methyl-l-propanesulfonic acid, or acrylic acid.

13. Improvement of claim 12 wherein the particles are about 100-300 μm in diameter and contain pores about 0.02 to 0.2 μm in diameter.

14. In the method of producing ribonucleotide diphosphates by phosphorolysis of polynucleotides in presence of immobilized polynucleotide phosphorylase, the improvement which comprises using polynucleotide phosphorylase immobilized to particles of a solid water-insoluble polymer of tris(hydroxymethyl)methylacrylamide.