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Engineered purine nucleoside phosphorylase variant enzymes

一种化嘌呤核苷磷酸化酶变体、化嘌呤核苷磷酸化酶的技术，应用在工程化嘌呤核苷磷酸化酶变体酶领域，能够解决核苷类似物化学复杂性等问题

Pending Publication Date: 2021-04-02
CODEXIS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Stalling of the enzyme causes premature termination of the DNA molecule, rendering it ineffective
However, generating nucleoside analogs by standard chemical synthesis techniques can be challenging due to their chemical complexity

Method used

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  • Engineered purine nucleoside phosphorylase variant enzymes
  • Engineered purine nucleoside phosphorylase variant enzymes
  • Engineered purine nucleoside phosphorylase variant enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0204] Preparation of wet cell pellets containing HTP-PNP

[0205] The parental gene for the PNP (SEQ ID NO:2) enzyme used to generate the variants of the present invention was obtained from the E. coli genome and cloned into the pCK110900 vector. W3110 E. coli cells were transformed with the corresponding plasmid containing the PNP-encoding gene, plated on LB agar plates containing 1% glucose and 30 μg / ml chloramphenicol (CAM), and grown overnight at 37°C. Pick a monoclonal colony and inoculate into 180 μl of LB containing 1% glucose and 30 μg / mL chloramphenicol, and place in a well of a 96-well shallow-well microtiter plate. Use the plate with O 2 A permeable seal was sealed and the culture was grown overnight at 30°C, 200 rpm and 85% humidity. Then, 10 μl of each cell culture was transferred to wells of a 96-well deep-well plate containing 390 μl TB and 30 μg / ml CAM. with O 2 Seal the deep well plate with a permeable seal and incubate at 30 °C, 250 rpm and 85% humidity ...

Embodiment 2

[0207] Preparation of cell lysates containing HTP PNP

[0208] Use 200 μl buffer containing 100 mM triethanolamine pH 7.5, 1 mg / mL lysozyme, 0.5 mg / mL PMBS and 5 mM MnCl 2 Lysis buffer prepared as described in Example 1 was used to lyse the cryoprecipitate. The cleavage mixture was shaken for 2 hours at room temperature. Plates were then centrifuged at 4000 rpm and 4°C for 15 min. The supernatants were then used as clarified lysates in biocatalytic reactions to determine activity levels.

Embodiment 3

[0210] Preparation of lyophilized lysates from shake flask (SF) cultures

[0211] Single colonies containing the desired gene picked from LB agar plates with 1% glucose and 30 μg / ml CAM and incubated overnight at 37°C were transferred to 6 ml LB with 1% glucose and 30 μg / ml CAM. Grow the culture at 30 °C, 250 rpm for 18 h, and subculture into 250 ml of TB containing 30 μg / ml CAM at about 1:50 to reach a final OD of about 0.05 600 . Grow the subculture at 30 °C, 250 rpm for approximately 195 min to reach an OD between 0.6-0.8 600 , and induced with 1 mMIPTG. Subcultures were then grown for 20 h at 30°C, 250 rpm. The subculture was centrifuged at 4000 rpm for 20 min. Discard the supernatant, and resuspend the pellet in 35 ml with 5 mM MnCl 2 25 mM triethanolamine buffer pH 7.5. use Cells were lysed by a processor system (Microfluidics) at 18,000 psi. The lysate was precipitated (10,000 rpm x 60 min), then the supernatant was frozen and lyophilized to produce a shake fla...

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Abstract

The present invention provides engineered purine nucleoside phosphorylase (PNP) enzymes, polypeptides having PNP activity, and polynucleotides encoding these enzymes, as well as vectors and host cellscomprising these polynucleotides and polypeptides. Methods for producing PNP enzymes are also provided. The present invention further provides compositions comprising the PNP enzymes and methods of using the engineered PNP enzymes. The present invention finds particular use in the production of pharmaceutical compounds.

Description

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 695,507, filed July 9, 2018, and U.S. Provisional Patent Application Serial No. 62 / 822,263, filed March 22, 2019, which are incorporated by reference for all purposes The two US provisional patent application series are hereby incorporated in their entirety. [0002] field of invention [0003] The present invention provides engineered purine nucleoside phosphorylase (purine nucleoside phosphorylase, PNP) enzymes, polypeptides with PNP activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing PNPases. The invention also provides compositions comprising PNP enzymes, and methods of using engineered PNP enzymes. The invention is especially useful in the production of pharmaceutical compounds. [0004] References to Sequence Listings, Tables or Computer Programs [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/45C12N9/00C12N9/10C12N15/52C12N15/54
CPCC12Y204/02001C12N9/1077C12N15/63
Inventor 斯科特·J·诺维克尼基·德拉斯韦丝娜·米切尔段达约瓦娜·纳佐尔奥斯卡·阿尔维左奥里克·安东尼·索厄尔-肯士杰弗里·C·穆尔马克·霍夫曼阿古斯蒂纳·罗德里格斯-格拉尼利奥迪帕克·维尔玛尼古拉斯·M·马歇尔杰伊·拉塞尔基思·A·卡纳达
Owner CODEXIS INC
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