Compositions of prokaryotic phenylalanine ammonia-lyase variants and methods of using compositions thereof
View Patent ↗Provided herein are phenylalanine ammonia-lyase (PAL) variants produced by prokaryotes, wherein such prokaryotic PAL variant has a greater phenylalanine-converting activity and/or a reduced immunogenicity as compared to a wild-type PAL. Further provided are compositions of prokaryotic PAL and biologically active fragments, mutants, variants or analogs thereof, as well as methods for the production, purification, formulation, and use of such compositions for industrial and therapeutic purposes, e.g., treating hyperphenylalaninemia, including phenylketonuria, and other disorders, including cancer.
1. A method for purifying an Anabaena variabilis phenylalanine ammonia-lyase (AvPAL) variant with minimal aggregation comprising:
(a) lysing bacterial cells containing the AvPAL variant by homogenization to generate a cell lysate;
(b) heating the cell lysate to 65° C. for 30 to 120 minutes;
(c) centrifuging the heated cell lysate, wherein a supernatant comprising the AvPAL variant is retained;
(d) filtering the supernatant to remove precipitates; and
(e) separating the AvPAL variant from contaminating proteins by sequential chromatography over an anion exchange (ALEX) column followed by a hydrophobic interaction (HIC) column, wherein the eluate from the HIC column comprises the AvPAL variant,
wherein the AvPAL variant has the amino acid sequence of SEQ ID NO: 11.
2. The method of claim 1 , further comprising:
(f) ultrafiltering or ultrafiltering/diafiltering the eluate from the HIC column comprising the AvPAL variant;
(g) pegylating the AvPAL variant by mixing polyethylene glycol with the AvPAL variant;
(h) removing free polyethylene glycol from the pegylated AvPAL variant by ultrafiltation/diafiltration; and
(i) formulating the pegylated AvPAL variant,
wherein said AvPAL variant comprises polyethylene glycol.
3. The method of claim 2 , wherein the ratio of AvPAL variant and the polyethylene glycol is about 1:3.
4. The method of claim 2 , wherein the AIEX column is a Toyopearl Giga Cap Q 650M column.
5. The method of claim 2 , wherein the HIC column is a Toyopearl Butyl 650M column.
6. The method of claim 2 , further comprising freezing and thawing the eluate from the HIC column comprising the AvPAL variant obtained in step (e), wherein one or more polyols or sugars selected from the group consisting of glycerol, sucrose, glucose, trehalose, mannitol and sorbitol are added to the HIC column eluate prior to freezing.
7. The method of claim 6 , wherein the polyol is glycerol.
8. The method of claim 7 , wherein the concentration of glycerol is 10% (v/v).
9. The method of claim 6 , wherein the sugar is sucrose.
10. The method of claim 9 , wherein the concentration of sucrose is 10% (v/v).
11. The method of claim 6 , wherein freezing the eluate from the HIC column comprising the AvPAL variant is performed in discrete temperature steps.
12. The method of claim 6 , wherein thawing the eluate from the HIC column comprising the AvPAL variant is performed in discrete temperature steps.
13. The method of claim 6 , wherein freezing and thawing the eluate from the HIC column comprising the AvPAL variant are performed in discrete temperature steps.
14. The method of claim 2 , wherein a diafiltration buffer in step (f) comprises potassium phosphate (KPi) and one or more agents consisting of trans-cinnamic acid (t-CA) and glycerol.
15. The method of claim 14 , wherein the diafiltration buffer comprises 50 mM KPi, 10 mM t-CA, 5% glycerol, pH 8.5.
16. The method of claim 2 , wherein a non-ionic detergent is added to the ultrafiltered or ultrafiltered/diafiltered eluate from the HIC column comprising the AvPAL variant obtained in step (f).
17. The method of claim 16 , wherein the non-ionic detergent is polysorbate 80 (PS80).
18. The method of claim 17 , wherein the concentration of PS80 is 0.02%.