FI63061C - STABILIZER THYMIDE DOSPHOSPHYL PREPARATION INNEHAOLLANDE RENAD TYMIDINPHOSPHORY WITH MEDICINAL PRODUCTS - Google Patents

Description

ΓΠβΕ ^ Π γβ! m1xAdvertisement, τη, Λ

JeBTq lBJ i11) UTLÄGGNINGSSKRIFT 6 3 0 6 1 • C (45) Fatei'i Lti.-Ν ':: .... lly 11 C4 1933 ^ (51) Kv.ik? /Int.ci.^ c 12 H 9/10, C o? G 7/00 ENGLISH I - FI N LAN D (21) PtMnttHmkemu · - PKmcina6linift «T9lU8l (22) H« kwnl «pUvi - Amflfcnlnpdm 09.05.79 ^ ^ ¢ 3) AlkupUvl — Glltlfh« ttdtf 27.01.76 (41) Tull iulklMlul - Mlvit offantllx pp qc; γρ

Patent- och ragisterstyralMn Anekin uti & gd och utlikrMtn pubiicond 31,12.82 (32) (33) (31) Requested etuellwM * —Begird prtoritM 27.01.75

England-England (GB) 3 * + ** 5/75 (71) The Wellcome Foundation Limited, 183-193 Euston Road, London NWl, England-England (GB) (72) Stanley Robert Morris Bushby, Durham, North Carolina, Thomas Anthony Krenitsky, Chapel Hill, North Carolina, USA (US) (7 * 0 Oy Kolster Ab (5 * 0 Stabilized thymidine phosphorylase preparation with improved thymidine phosphorylase for improved storage stability - Stabiliserat thymidine phosphorylase preparations The present invention relates to a stabilized, purified thymidine phosphorylase preparation with improved storage stability, which has been isolated from application 760181 (patent 56856) -Avdelad frän trsökan 76OI8I (patent 56856).

The preparation according to the invention is characterized in that it consists of a concentrated aqueous solution of thymidine phosphorylase containing at least 5 mg / ml of protein in an ammonium sulphate-containing phosphate buffer as a first step, adsorption is performed on a calcium phosphate gel containing equivalent amounts of calcium and phosphate ions, followed by chromatography and dialysis steps.

Thymidine phosphorylase has previously been purified from bacteria such as Salmonella typhimurium, Bacillus cereus, Bacillus stearothermo-philus and Haempophilus influenzae, and in particular from Escherishia coli, which requires 63061 thymines and netionin for growth. The latter purification requires a very lengthy process involving precipitation, fractionation, several chromatographic steps and dialysis (Schwartz, M, 1971 »Eur. J. Biochem. 21: 191-198). However, the enzyme preparation obtained by this method was only 25-fold purer than the crude cell extract.

It has recently been found that certain bacterial strains, especially a certain E. coli strain, produce unusually high levels of thymidine phosphorylase under suitable growth conditions, and that this enzyme can be isolated and purified by applying the cell extract to certain adsorbents and eluting with much higher purity. than what has been possible so far. In addition, this new isolation / purification method can be applied to the large-scale production of this enzyme.

The preparation of purified thymidine phosphorylase of bacterial origin thus comprises the following steps: extraction of the crude enzyme from the bacteria into an aqueous medium, fractionation of the extract, comprising chromatography and dialysis steps. This method is characterized by 1) selecting a bacterial strain capable of producing high levels of thymidine phosphorylase under suitable growth conditions, and 2) applying the crude cell extract obtained as a first purification step to a calcium phosphate gel absorbent containing substantially equivalent amounts of Ca ++ and PO ++. The eluent is preferably then contacted with diethylaminoethylcellulose (DEAE-cellulose) and / or cellulose-epichlorohydrin triethanolamine (ECTEOLA-cellulose), and after elution from DEAE-cellulose and before adsorption on ECTEOLA-cellulose is dialyzed against water or dialyzed against water.

Escherichia coli B-96 (ATCC 13-73) is remarkably preferred for use in the purposes of the invention. An example of another bacterial strain that produces large amounts of thymidine phosphorylase and is thus suitable for use in the present invention is Salmonella typhimurium LT-2 (ATCC 15277). In some cases, when a more thermally stable enzyme is desired, an enzyme isolated from B. stearothermophilus has proven to be effective.

E. coli strain ATCC 13-73 can be cultured in a minimal salt medium containing a suitable carbon source and in addition purines. Alternatively, the bacterium is cultured in a yeast extract medium. The crude extract of the enzyme can then be prepared by sonication of the bacterial cells in phosphate buffer, after which the mixture is centrifuged to remove cell debris.

3 63061

For example, the crude extract is mixed with a small amount of calcium phosphate gel, and the mixture is centrifuged to remove unwanted proteins. The supernatant thus obtained can then be mixed with another batch of calcium phosphate gel, whereupon the enzyme is absorbed therein. Enzyme activity can be eluted from the gel by serial washing with phosphate buffer. The enzyme can then be adsorbed onto DEAE cellulose, washed and eluted therefrom. After dialysis, the product can be adsorbed onto and eluted from ECTEOLA cellulose.

To monitor elution, enzyme activity is measured at all purification steps as appropriate using spectrophotometric measurement at the appropriate wavelength.

The purified enzyme thus obtained can be suitably provided as a suspension in aqueous ammonium sulfate solution.

One unit of enzyme activity of the purified enzyme is the amount of enzyme that catalyzes the formation of one mole of thymine per minute from a 1 mM solution of thymidine at 25 ° C in the presence of 200 mM potassium phosphate buffer at pH 7.

The enzyme is also present in many vertebrate tissues from which it can be prepared pure, but enzyme levels in mammalian tissues are generally much lower than those of bacteria. In addition, purified thymidine phosphorylase of bacterial origin is often more active than the corresponding purified mammalian.

Horse blood has been found to contain 00-100 units of thymidine phosphorylase activity (as defined herein) per ml. The E. coli ultrasonic digestion product of Example 1 contains more than 1,000 times this concentration. Thus, the advantages of an economical method of producing a purified enzyme prepared from a bacterial source are many and significant. Not only is it a question of a more readily available, inexpensive source of enzyme, but also a method for extracting and purifying the enzyme is much more economical.

It is already known that thymidine phosphorylase of bacterial origin is stable at -20 ° C, but that at 1 + ° C the activity decreases at a considerable rate. The thymidine phosphorylase preparation according to the invention is considerably more stable due to its ammonium sulphate, provided that the protein content of the preparation is at least 5 mg / ml. Concentrated solutions of the enzyme (5 mg / protein / ml or more) in phosphate buffer containing 10% ammonium sulphate can be used, for example. periods without a decrease in activity or with a slight decrease in activity. Permanent suspensions of thymidine phosphorylase in aqueous ammonium sulfate can also be prepared.

it

The following example illustrates the invention. 63061

Example

Purification of thymidine phosphorylase E. coli B-96 (ATCC 13-73) strain was grown in air-conditioned vessels at 3 ° C in minimal salt medium containing Na 2 HPO 2 (18.9 g / l), KH PO 2 (6.3 g / l) ), MgSO 4 .H 2 O (0.2 g / l), (NH 4 J 2 SO 2 (2.0 g / l), adenosine (0.5 g / l) and g / l), When the optical density of the culture reached 2.0 at 600 nm (without dilution), the cells were harvested by centrifugation, and the following procedures were performed at 3 ° C, unless otherwise indicated: The cell paste (25 g) was suspended in its own weight. to a double volume of 5 mM potassium phosphate buffer, pH 8.0 (Buffer A) 5 ml aliquots of the cell suspension were each sonicated for two 12-second periods with cooling for 50 seconds between treatments, using a Branson Model 5125 Sonic Oscillator with a power reading of 6. Ultrasonic combined and centrifuged for 20 minutes at k8,000 xg.

To the supernatant (Step I - see Table I below) was slowly added 25 mL of calcium phosphate gel suspension (31 mg of dry solid per mL, aged at 3 ° C for 5 months). The resulting suspension was stirred for 10 minutes, then centrifuged at 12,000 x g for 5 minutes. The supernatant was again mixed with the calcium phosphate gel suspension (100 ml), and the mixture was stirred for 10 minutes and centrifuged at 97,000 x g for 15 minutes. The resulting gel precipitate was washed with buffer A (100 ml) by resuspension and centrifugation at 97,000 x g for 15 minutes.

Enzyme activity was extracted from the gel precipitate by two successive washes: first with 10 mM potassium phosphate buffer, pH 8.0 (100 ml) and then with 20 mM potassium phosphate buffer, pH 8.0 (100 ml). Both broths were combined (Step II).

The remainder of the preparation was performed at 25 ° C. The combined washings were applied to a DEAE cellulose column, 1.8 cm in diameter, 6 cm high, pre-equilibrated with 20 mM potassium phosphate, pH 6.1+ (buffer B). The packed column was washed with buffer B (100 ml), and the enzyme was then eluted with a linear gradient of phosphate buffer. This gradient was prepared by carefully adding, with stirring, 200 mM potassium phosphate buffer, pH 6, U (200 mL) to a mixing vessel originally filled with buffer B (200 mL) at such a rate that a constant volume remained in the mixing vessel throughout. Fractions with the highest enzyme activity were pooled (step lii) and dialyzed against water 63061 (2 L) for 1 hour using a cellulose dialysis tube (8 mm diameter). The dialysis was repeated and then the dialysate was applied to an ECTEOLA cellulose column (1.8 x 5 cm) pre-equilibrated with buffer A. The packed column was washed with buffer A (100 ml). The enzyme was then eluted with a linear gradient of eluent prepared as above using a mixing vessel originally filled with buffer A (200 ml) and siphoned with 200 mM potassium phosphate buffer, pH 8.0 (200 ml) by gravity. as the column solution progressed. Fractions containing enzyme activity were pooled (step IV), and sufficient ammonium sulfate was added to provide a suspension of the enzyme in an 80- $ ammonium sulfate solution. This method resulted in a total 100-fold increase in specific activity for the protein and a substantially complete elimination of nucleic acids.

Thymidine phosphorylase activity was tested by spectrophotometrically measuring the decrease in absorbance at 290 nm after the conversion of thymidine to thymidine by thymidine phosphorylase. At 290 nm pH 7, thymidine has a higher extinction coefficient than thymine (Δ £ = -ί * 8θ M 1 cm 1). The test mixture contained 200 mM potassium phosphate buffer, pH 7, and 1 mM thymidine, for a total volume of 2.5 ml. One unit of enzyme activity is the amount that catalyzes the formation of one mole of thyrine from thymidine in 1 minute at 25 ° C.

The results of the purification described in this example are summarized in Table I. The method has been performed in 60-fold amounts with similar results.

6 63061 co ίΐ s s s •• 3 -S .5 .5

-5 I f0 rö <xJ

P -P Ή P

® aJ

x x x i, i i

SH O i — I

o ^ ^ S

.s § §5 5 O CM O 00 m dP o oo co .3 · tn w 1-1

•B

Cu il + -> .S> -P I: CT £ O o o o

rn -H -μ · Η -μ ft 1-0 CO O O

ΰ μ φ ι / ι · Η cd οο ο οο

3 X ® Χ ·· 0 W

35 <4-> >> - y CM Γ 'CM CD

'P CNJ 00 CO., LTD

& Λ ~ P · Η ΰ Φ Η σι rH σ>

i2 P S a> (O rH

"tj 0 ^ ·>»> n 3 Päg? o o o o

•B

T3 "£>: CT £ oooo 4-1 ro oooo

-.- H ro o co o CM

W H: θ H λ λ »» f,

^ ίξ ^ CO LO CD CO

Jtoocoj * 3 Jj W · Ή en co h co

5 ° ^ CD * * ^ O

H-5 ω

•B

: S

CT I I OOOO

Ä> Η · Η oooo (-L ·> 4-> 1: cö o co lo t'- _ (Ή P 'HP rr> rt Λ JS ή Φ ω * h H co r ~ oo + r, χ <D Pc: θ E o 01 00 co

“<C P>, X rH

C/O

CD

CQ

s ai

u · Η w LO CO CM CO

H co o lo co • 3 <x

1 I

ΐΐ CT M M M>

S> M H M

W M

dl R itJ

P rH I: ct tn

(fl 4) g P I Q

Φ -μ 9 in CT O

C -μ · η -h tn H

• H iiri n 9 CT en rH Φ o r-1

>: CT CT Φ H rH

ω 32 32 W) 9 0): CT 1 — I · Ρ · Η rH tn

E O C · Η P rH · Η I, "P

rP: CTdPPCTP <CP

0): CT: CT Φ -P CT tn P -1 P>

4-i rH CT Φ CTCT I CT O CT

2j 1¾ ä8 öS

J3 ^ OHiCTHOyHOH

Ä. di d · π: j Uh q φ 14 <u