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Compositions of prokaryotic phenylalanine ammonia-lyase variants and methods of using compositions thereof

A technology of algae phenylalanine ammonia lyase and variants, applied to the composition of prokaryotic phenylalanine ammonia lyase variants and the field of using its composition, can solve the lack of structural and biochemical knowledge, and have not yet coordinated issues such as concerted efforts

Inactive Publication Date: 2012-10-24
BIOMARIN PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no concerted effort aimed at improving these parameters due to lack of structural and biochemical knowledge about this protein

Method used

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  • Compositions of prokaryotic phenylalanine ammonia-lyase variants and methods of using compositions thereof
  • Compositions of prokaryotic phenylalanine ammonia-lyase variants and methods of using compositions thereof
  • Compositions of prokaryotic phenylalanine ammonia-lyase variants and methods of using compositions thereof

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Experimental program
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Embodiment approach

[0258] Also provided herein is a method of purifying prokaryotic PAL or a biologically active fragment, mutant, variant or analog thereof. According to an exemplary first embodiment, the transformed cell mass is grown and disrupted, leaving behind the crude recombinase. Extraneous material is usually separated from the crude block to prevent contamination of the column. Utilize one or more chromatographic resins for chromatographic purification. Next, the purified protein is formulated into a buffer designed to provide stable activity over an extended period of time. In another embodiment, the method of purifying prokaryotic PAL, or a biologically active fragment, mutant, variant or the like thereof, comprises: (a) utilizing a pressure homogenizer (but potentially, by other physical means, such as glass bead lysis) to lyse bacteria containing recombinant prokaryotic PAL or its biologically active fragments, mutants, variants, or the like; (b) heat treatment; (c) using a seco...

Embodiment 1

[0262] Cloning of Nostoc punctatus and Anabaena variabilis PAL

[0263] DNA manipulation

[0264] Genomic DNA of Nostoc punctatus was purchased from ATCC (29133D) and the PAL gene (ZP_00105927) was amplified by PCR with primers 5'-CACTGTCATATGAATAAACATCTCTACAACAGAACAT-3' (SEQ ID NO: 12) and 5'-GACAGTGGCGGCCGCTCACGTTGACTTTAAGCTCGAAAAAATATG-3' (SEQ ID NO: 13) increase. The resulting PCR product was digested with NdeI and NotI and the 1.7kb fragment was ligated into pET-28a(+) and pET-30a(+) (Novagen) with and without N-His tagging, respectively .

[0265] Anabaena variabilis cells were purchased from ATCC (29413). Genomic DNA was extracted (Qiagen) and the PAL gene (YP_324488) was amplified by SOE-PCR to remove the NheI site. Primer 1 (5'-CACTGTGCTAGCATGAAGACACTATTCTCAAGCACAAAG-3') (SEQ ID NO: 14) and primer 2 (5'-GGAAATTTCCTCCATGATAGCTGGCTTGGTTATCAACATCAATTAGTGG-3') (SEQ ID NO: 15) were used to amplify nucleotides 1-1190 and primer Primer 3 (5'-CCACTAATTGATGTTGATAACCAAGCCA...

Embodiment 2

[0272] Purification of NpPAL and AvPAL

[0273]The culture was centrifuged at 5,000 g for 20 min in a tabletop centrifuge and the supernatant was discarded. Cell pellets are usually frozen at –70°C prior to further processing. After thawing, the pelleted cells were suspended to approximately 80 optical density units (600 nm) in TBS (25 mM Tris, 150 mM NaCl, pH 7.8). Cells were lysed by passing through the APV pressure homogenizer twice at 12-14,000 psi. The crude lysate was then heat treated at 55 °C for 2 h. The lysate was centrifuged at 10,000 g for 30 min, and the supernatant was retained and filtered with a 0.2 μm vacuum filter (Corning).

[0274] PAL was purified from the clarified lysate by passing it sequentially through a Butyl 650M column (Tosoh BioSciences) followed by a MacroPrep High Q column (BioRad). The products eluted by both methods of SDS PAGE and reverse phase HPLC showed high purity.

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Abstract

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).

Description

technical field [0001] Provided herein are compositions related to prokaryotic phenylalanine ammonia lyase (PAL) variants, including optimizing such compositions to increase prokaryotic PAL catalytic activity and / or stability while reducing prokaryotic PAL immunogenicity and / or protein Hydrolysis sensitivity. Also provided herein are such optimal compositions of prokaryotic PAL variants in therapeutic (e.g., treatment of hyperphenylalaninemia (HPA), including phenylketonuria (PKU); and other diseases, including cancer )the use of. Background of the invention [0002] Phenylalanine ammonia lyase (PAL) is a non-mammalian enzyme widely distributed in plants (Koukol, et al., J. Biol. Chem. 236:2692-2698 (1961); Hanson, et al., The Enzymes 7:75-166 (1972); Poppe, et al., Curr.Org.Chem.7:1297-1315 (2003)), some fungi (Rao, et al., Can.J.Biochem.4512:1863-1872 (1967); Abell, et al., Methods Enzymol.142:242-253 (1987)) and bacteria (Bezanson, et al., Can.J.Microbiol.16:147-151 (1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/00
CPCA61K47/183C12Y403/01024C12N9/88A61K47/48215A61K47/10A61K38/51C12N9/96A61K9/0019A61K47/60A61P3/00A61P35/00A61P35/02A61P7/00C07K1/14
Inventor 奥古斯塔斯·O·奥克哈玛菲肖恩·M·贝尔G·尼克·泽切尔勒克里斯·安东森张彦宏基尤·Y·莱P·A·菲茨帕特里克E·D·卡基斯M·C·维拉德丹尼尔·J·温特穆巴拉克·穆塔利夫
Owner BIOMARIN PHARMA INC
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