假单胞菌表达重组人干扰素α-2b蛋白纯化用离子交换树脂的筛选  

Screening of ion -exchange resins for purification of recombinant human interferon α-2b expression by Pseudomonas putida

作　　者：梁士民[1] 钱晓露[1] 范凯[1] 闫凤英[1] 

机构地区：[1]天津华立达生物工程有限公司,天津300457

出　　处：《生物医学工程与临床》2012年第5期425-429,共5页Biomedical Engineering and Clinical Medicine

基　　金：天津市滨海新区高新技术企业培育资金项目(2010-BK-200112)

摘　　要：目的研究用于分离纯化假单胞菌表达重组干扰素α-2b蛋白的离子交换树脂。方法利用工程菌假单胞菌发酵获得的菌体进行裂解,经离心、沉淀、盐析、疏水层析、超滤、透析等步骤,获得重组人干扰素α-2b蛋白溶液,通过对比8种商品化离子交换树脂(DE 52、DEAE FF、Fractogel DEAE、Bio-sep DEAE、CM 52、CM FF、Fractogel COO-、Bio-sep CM)的动态载量、洗脱条件及纯化后的重组人干扰素α-2b蛋白的活性回收率,采用安捷伦2100生物分析仪比较纯化后的重组人干扰素α-2b纯度,考察离子交换树脂用于假单胞菌表达重组干扰素α-2b蛋白分离纯化的适用性。结果 FractogelDEAE和Fractogel COO-的动态载量最高,分别为183.04 mg/mL和107.00 mg/mL;Fractogel DEAE和CM 52洗脱效果较好;DEAE FF和CM FF纯化后的干扰素α-2b蛋白活性回收率较高,分别为82.38%和49.46%。结论 DEAE FF和CM FF适用于假单胞菌重组干扰素α-2b蛋白的分离纯化。Objective To study the screening of ion-exchange resins used for purification of recombinant human interferon α-2b expression by Pseudomonas putidaa, putida）. Methods The p.putida biomass harvested from fermentation were extracted, and acquired recombinant human interferon α -2b solution by steps of centrifuge, precipitation, ammonium sulfate precipitation, hydrophobic interaction chromatography, ultra filtration and dialysis. The dynamic loading, elution condition and yield of recombinant human interferon α-2b in 8 different resins （DE 52, DEAE FF, Fractogel DEAE, Bio-sep DEAE, CM 52, CM FF, Fractogel COO- and Bio-sep CM） were compared. After purification, the Agilent 2100 bioanalyzer was used to compare purity of recombinant human interferon α-2b after purification, and investigated the applicability of ion-exchange resin for isolation and purification of recombinant human interferon α-2b expression by p. putida. Results Fractogel DEAE and Fractogel COO- had larger dynamic loading of 183.04 mg/mL and 107.00 mg/mL, respectively. Fractogel DEAE and CM 52 had better elution resolution, the DEAE FF and CM FF had higher yield for purified interferon α-2b at 82.38 % and 49.46 %, respectively. Conclusion It is demonstrated that DEAE FF and CM FF are more suitable for purification of recombinant human interferon α-2b expressed by p. p utida.

关 键 词：离子交换树脂 重组干扰素Α-2B 动态载量 洗脱条件 蛋白纯化 

分 类 号：R943[医药卫生—药剂学]