重组Tβ4②分离纯化条件的优化及实验室研究工艺的确定  

Optimization of Purification Conditions for Recombinant Tβ4②and the Determination of Preparation Technique in Laboratory

作　　者：张雨婷 柳佳欣 何磊 张阔 王舒宁 郝强 李萌 张旺倩 ZHANG Yu-ting;LIU Jia-xin;HE Lei;ZHANG Kuo;WANG Shu-ning;HAO Qiang;LI Meng;ZHANG Wang-qian(State Key Laboratory of Cancer Biology Shanxi Province,Xi'an 710032,China;Department of Biopharma-ceutics,School of Pharmacy,Air Force Medical University,Xi'an 710032,China;School of Basic Medical Sciences,Air Force Medical University,Xi'an 710032,China)

机构地区：[1]肿瘤生物学国家重点实验室,陕西西安710032 [2]空军军医大学药学系生物制药学教研室,陕西西安710032 [3]空军军医大学学员二大队八队,陕西西安710032

出　　处：《药物生物技术》2021年第3期239-244,共6页Pharmaceutical Biotechnology

基　　金：陕西省重点研发计划(No.2019SF-087)。

摘　　要：优化重组蛋白Tβ4②的纯化条件,最终获得纯度大于98%的具有生物活性的Tβ4②,为后续研究奠定基础。采用单因素试验,研究裂解液体积、裂菌方法、超声功率、硫酸铵饱和度等对重组蛋白纯化的影响,确定最佳的裂解方法和硫酸铵饱和度;通过对Tβ4②蛋白理化性质的预测,确定最佳的层析方案。采用1∶7的菌体与裂解液体积比,45%的超声功率或高压匀浆技术,50%的硫酸铵饱和度作为粗提纯的最佳条件,并通过疏水作用层析分离获得了纯度大于98%的重组蛋白。该研究优化了重组蛋白Tβ4②的纯化条件,最终获得了纯度大于98%的重组蛋白,蛋白回收率为(0.26±0.02)%,且与Tβ4单体相比,Tβ4②可以明显促进内皮细胞的迁移。Thymosin beta 4(Tβ4)is a peptide with 43 amino acids that is critical for repair and remodeling tissues on the skin,eye,heart,and neural system following injury.However,it cannot be expressed in E.coli directly because of its low molecular weight.In the previous study,the authors have constructed recombinant genetically engineered bacteria p ET22 b(+)-Tβ4②/BL21(DE3)and induced with IPTG.The results showed that Tβ4②is able to promote lymphocyte differentiation and proliferation as effectively as synthetic one,and has weak immunogenicity detected by ELISA.Based on the optimization of the fermentation conditions of p ET22 b-Tβ4②/BL21(DE3),the final bacterial yield of p ET22 b-Tβ4②/BL21(DE3)and the expression level of Tβ4②were all reached to the highest level,with the yield and protein expression were 49 g/L and 30%,respectively.However,there are various conditions and factors affecting subsequent protein separation and purification,including protein expression level,cell fragmentation effect,and physicochemical properties of proteins,etc.So far,there was no single or a set of ready-made methods to extract any protein from complex mixtures.To optimize the purification conditions of recombinant Tβ4②,and finally obtain a recombinant protein with a purity of greater than 98%,laying a foundation for in vitro research,on the one hand,the authors used single-factor experiments to study the effects of lysate volume,cleavage method,ultrasonic power,ammonium sulfate saturation,etc.On the cleavage effect and crude purification effect to determine the best crude separation conditions.On the other hand,the authors use software to predict the physical and chemical properties of Tβ4②and determine the best chromatography scheme.Rough separation conditions:1∶7 volume ratio of bacteria to lysate,45%ultrasonic power or high-pressure homogenization technique,50%ammonium sulfate saturation;fine separation conditions:hydrophobic interaction chromatography.This study optimized the purification conditions of recombi

关 键 词：胸腺肽β4 二串体 大肠杆菌 细胞破碎 盐析 疏水层析 

分 类 号：TQ93[化学工程]