PEGylation of yeast cytosine deaminase for pretargeting.
PEGylation of yeast cytosine deaminase for pretargeting.
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用于预靶向的酵母胞嘧啶脱氨酶的聚乙二醇化。
DOI：
10.1002/jps.20354
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Kwon,GlenS
中科院分区：
文献类型：
--
作者：
Xiong,MayP;Kwon,GlenS
酵母胞嘧啶脱氨酶（yCD）的克隆，表达，并通过亲和层析纯化。我们已经表征了由2-4个PEG链共价连接在yCD上产生的产物，并确定了每种相应缀合物的主要和次要异构体。结果表明，对于非共价缔合的同源二聚体，可以通过同时使用尺寸排阻色谱（SEC）、MALDI-TOF MS和SDS-PAGE凝胶来表征和推断单个亚基上的PEG化水平。结果还表明，与我们预期的相反，将两个以上的PEG链连接到yCD降低了其稳定性。酶活性研究表明，在yCD上融合N-末端纯化标签对5-氟胞嘧啶或胞嘧啶亲和力没有显著影响，表观周转率保持在105 M −1· s−1以内。在37°C下的稳定性研究表明，对于yCD和2 mPEG 5 K-yCD，t1/2= 8-9 h，而对于3-、4 mPEG 5 K-yCD和yCD/BSA，t1/2< 2 h。BSA与yCD的孵育也降低了在37°C下长时间孵育的酶稳定性。如果yCD用于预靶向策略，则该发现是重要的。© 2005 Wiley-Liss公司。和美国药剂师协会
Yeast cytosine deaminase (yCD) was cloned, expressed, and purified by affinity chromatography. We have characterized the products resulting from covalent attachment of 2–4 PEG chains on yCD and determined the major and minor isomers for each respective conjugate. The results show that for non-covalently associated homodimers, it is possible to characterize and deduce PEGylation levels on individual subunits through the concurrent use of size exclusion chromatography (SEC), MALDI-TOF MS, and SDS–PAGE gels. The results also show that contrary to what we expected, attaching more than two PEG chains to yCD decreased its stability. Enzymatic activity studies revealed that the fusion of an N-terminus purification tag on yCD has no significant effect on 5-fluorocytosine or cytosine affinity, with apparent turnover rates remaining within 105M−1· s−1. Stability studies at 37°C revealed thatt1/2= 8–9 h for yCD and 2mPEG5K-yCD, whereas for 3-, 4mPEG5K-yCD and yCD/BSA, t1/2< 2 h. Incubation of BSA with yCD also decreased enzyme stability over prolonged incubation at 37°C. This finding is important if yCD is to be used in a pretargeting strategy. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association