亲和标签在重组蛋白表达与纯化中的应用

亲和标签融合技术为重组蛋白的纯化提供了一种简单方便的纯化工具,具有结合特异性高、洗脱条件温和、通用性强、纯化倍数高等显著优点。概述了亲和标签对融合蛋白表达的影响,可以提高重组蛋白的产量,增强重组蛋白的可溶性,促进重组蛋白的正确折叠;回顾了在重组蛋白表达与纯化中广泛使用的几种亲和标签,以及近年来相继出现的几种比较新颖的纯化标签;介绍了亲和标签的组合使用策略,His6-MBP组合标签集合了两个标签的优点,串联亲和纯化可以纯化获得生理条件下的蛋白质复合体;展望了亲和标签未来的发展趋势,认为仍需继续开发性能更加优越、纯化效果更加显著的纯化标签系统。     

FLAG融合短肽在重组蛋白质纯化中的应用

FLAG融合标签由八个氨基酸组成(AspTyrLysAspAspAspAspLys),专门设计用于重组蛋白质的免疫吸附纯化。现已发展出了多个针对该短肽的抗体,包括M1、M2和M5单克隆抗体。FLAG标签与融合的重组蛋白质之间一般含有一个肠激酶切割位点,可以用肠激酶切割除去。FLAG融合标签技术具有简单、快速进行定量检测的优点,在重组蛋白质的分离纯化中应用广泛。     

猪型布鲁氏菌rOmp3148-74-BLS融合肽的诱导表达与纯化

目的:为了研究布鲁氏菌Omp31分子的抗原性质,以及布鲁氏菌BLS蛋白质的聚合功能,通过融合PCR技术,制备布鲁氏菌Omp3148-74与BLS的融合蛋白质,并且进行蛋白质的纯化。方法:以猪型布鲁氏菌Omp31基因和BLS基因作为研究对象,通过融合PCR技术构建重组表达载体,IPTG诱导融合蛋白质表达之后,利用His-tag亲和层析柱进行亲和纯化。结果:Omp3148-74-BLS融合DNA长度为531 bp,编码177个氨基酸;加上原核表达载体上的His-tag标签,融合蛋白质的实际大小为25.07 kD,实验结果证实表达载体构建正确,重组表达正确。结论:通过融合PCR技术构建的重组子pET-28-a-Omp3148-74-BLS,可以在大肠杆菌中成功表达;His-tag亲和层析技术可以纯化到Omp3148-74-BLS融合蛋白,而且蛋白质纯度较高。     

重组蛋白质亲和标签的选择与串联亲和纯化的应用进展

重组蛋白质技术作为蛋白质研究的重要手段之一,在生物化学与生物物理学研究领域,扮演着极其重要的角色.亲和纯化作为最为方便与快捷的重组蛋白质纯化手段,日益得到广泛的应用.由于各种亲和标签,纯化介质层出不穷,性质各异.应根据自身研究对象具体情况选择合适的亲和纯化标签.近年双亲和标签进行串联亲和纯化日益成为蛋白质相互作用研究的重要方法.多种亲和标签的搭配已得到成功应用,部分已进入商业化,并在各种模式生物中得到广泛的应用,本文就重组蛋白质亲和标签的选择与串联亲和纯化作一综述.     

重组标签蛋白在蛋白质纯化中的研究进展

重组蛋白的表达纯化是研究蛋白质结构与功能的重要环节之一,表达重组蛋白宿主细胞的监测筛选和重组蛋白低水平可溶性表达、低回收率以及不稳定易水解等问题一直是蛋白质高通量纯化工艺中的难题。近年来,将多肽或蛋白质作为标签,与目标蛋白共表达的方法已经很大程度地解决了这一问题。因此,针对不同特性的蛋白质选择合适的标签纯化策略在重组蛋白纯化研究中是相当关键的环节。本文回顾了几种传统标签蛋白的研究概况(His-tag,Arg-tag,Flag-tag,GST-tag,MBP-tag等),着重对近年来新开发的了几种标签蛋白(Si-tag,Halo-tag,Intein-CBD-tag,ELPs-tag等)进行了深入探讨,并对新标签蛋白在蛋白质纯化中的应用前景作以展望。     

重组蛋白质纯化中切割蛋白酶的选择与应用进展

现代生物医药的发展越来越依赖大分子药物的开发与应用,其中蛋白质药物所占的比重越来越高,生产需求进一步加大.重组蛋白质技术作为一种方便的蛋白质生产来源,日益得到广泛运用.重组蛋白质纯化过程中往往使用到融合亲和纯化标签,然而这些冗余的标签在蛋白质精细化生产中,尤其是作为蛋白质医药存在时,是必须被予以去除的.切割蛋白酶发挥着不可替代的作用.本文就现有的各种常用切割蛋白酶进行比较和总结,为重组蛋白质生产与科研者提供参考.     

多聚组氨酸融合标签在蛋白药物开发中的应用

纯化技术是制约蛋白质药物开发及其产业化的关键技术之一。构建多聚组氨酸标签融合蛋白,采用固定金属离子亲和层析进行纯化,是一种高效的蛋白质纯化策略。介绍多聚组氨酸融合标签在蛋白质药物开发中的应用基础和应用概况,分析多聚组氨酸标签在融合蛋白中的位置对亲和层析纯化的影响,总结常用的多聚组氨酸融合表达方式,并对其融合表达样品的预处理、亲和层析纯化条件及其对目的蛋白药物药用安全性和有效性的影响进行探讨。     

利用EDA融合标签高效表达猪圆环病毒2型壳蛋白

原核生物作为宿主细胞被广泛应用于异源蛋白质的重组表达,并且为生物活性蛋白质的制备提供了一种高效、经济的方法,因而在分子生物学中得到普遍的应用。然而,病毒蛋白在使用原核重组表达系统进行重组表达时,会出现病毒蛋白溶解性差和表达量低等问题。因此,通过使用各种融合标签以增加目的重组蛋白的表达量和溶解性成为有效的方法。本研究通过使用3种融合标签（EDA标签、MBP标签和GST标签）以获得表达量高的可溶性重组表达猪圆环病毒2型壳蛋白;并比较3种融合标签对该蛋白表达量、溶解性和稳定性的影响。研究结果表明,EDA标签可以显著提高重组表达的猪圆环病毒2型壳蛋白表达量,并且能够增强该蛋白的稳定性;MBP标签可增强重组表达的猪圆环病毒2型壳蛋白表达量,但是不能改善该蛋白的稳定性;GST标签能够增强该重组表达蛋白的表达量,但是不能增强该蛋白的溶解性和稳定性。本研究将EDA作为PCV2-CP蛋白的融合标签,显著提高PCV2-CP-EDA重组蛋白的表达量和增强该重组蛋白的稳定性,为病毒蛋白的可溶性重组表达提供了一种新的融合标签。     

利用EDA融合标签高效表达猪圆环病毒2型壳蛋白（英文）

原核生物作为宿主细胞被广泛应用于异源蛋白质的重组表达,并且为生物活性蛋白质的制备提供了一种高效、经济的方法,因而在分子生物学中得到普遍的应用。然而,病毒蛋白在使用原核重组表达系统进行重组表达时,会出现病毒蛋白溶解性差和表达量低等问题。因此,通过使用各种融合标签以增加目的重组蛋白的表达量和溶解性成为有效的方法。本研究通过使用3种融合标签(EDA标签、MBP标签和GST标签)以获得表达量高的可溶性重组表达猪圆环病毒2型壳蛋白;并比较3种融合标签对该蛋白表达量、溶解性和稳定性的影响。研究结果表明,EDA标签可以显著提高重组表达的猪圆环病毒2型壳蛋白表达量,并且能够增强该蛋白的稳定性;MBP标签可增强重组表达的猪圆环病毒2型壳蛋白表达量,但是不能改善该蛋白的稳定性;GST标签能够增强该重组表达蛋白的表达量,但是不能增强该蛋白的溶解性和稳定性。本研究将EDA作为PCV2-CP蛋白的融合标签,显著提高PCV2-CP-EDA重组蛋白的表达量和增强该重组蛋白的稳定性,为病毒蛋白的可溶性重组表达提供了一种新的融合标签。     

GST-HRB融合蛋白的表达与纯化

构建GST-HRB重组质粒,进行融合蛋白的表达、纯化及鉴定.利用PCR扩增及基因重组技术,以pcDNA-3.1-HRB为模板扩增出HRB全基因序列,并将其插入带有GST(谷胱甘肽巯基转移酶)标签的原核表达载体pGEX-6P-1中,构建GST-HRB融合蛋白表达质粒.然后,将重组质粒GST-HRB转化至大肠杆菌Rosseta进行融合蛋白的表达.利用GST琼脂糖珠进行融合蛋白的纯化,最后应用SDS-PAGE电泳和Western blotting鉴定纯化的融合蛋白.结果表明,成功构建pGEX-6P-1-HRB原核表达载体,表达及纯化了GST-HRB融合蛋白.     

Mocr: a novel fusion tag for enhancing solubility that is compatible with structural biology applications

A persistent problem in heterologous protein production is insolubility of the target protein when expressed to high level in the host cell. A widely employed strategy for overcoming this problem is the use of fusion tags. The best fusion tags promote solubility, may function as purification handles and either do not interfere with downstream applications or may be removed from the passenger protein preparation. A novel fusion tag is identified that meets these criteria. This fusion tag is a monomeric mutant of the Ocr protein (0.3 gene product) of bacteriophage T7. This fusion tag displays solubilizing activity with a variety of different passenger proteins. We show that it may be used as a purification handle similar to other fusion tags. Its small size and compact structure are compatible with its use in downstream applications of the passenger protein or it may be removed and purified away from the passenger protein. The use of monomeric Ocr (Mocr) as a complement to other fusion tags such as maltose-binding protein will provide greater flexibility in protein production and processing for a wide variety of protein applications.     

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.     

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.     

Self-cleaving fusion tags for recombinant protein production

Fusion expression is a common practice for recombinant protein production. Some fusion tags confer solubility on the target protein whereas others provide affinity handles that facilitate purification. However, the tag usually needs to be removed from the final product, which involves using expensive proteases or hazardous chemicals and requires additional chromatography steps. Self-cleaving tags are a special group of fusion tags that possess inducible proteolytic activity. Combined with appropriate affinity tags, they enable fusion purification, cleavage and target separation to be achieved in a single step, which saves time, labor and cost. This paper reviews currently available self-cleaving fusion tags for recombinant protein production. For each system, an introduction of its key characteristics and a brief discussion of its advantages and disadvantages is given.     

Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions

Immobilized metal affinity chromatography (IMAC) of proteins containing poly-histidine fusion tags is an efficient research tool for purifying recombinant proteins from crude cellular feedstocks at laboratory scale. Nevertheless, to achieve successful purification of large amounts of the target protein for critical therapeutic applications that demand the precise removal of fusion tags, it is important to also take into consideration issues such as protein quality, efficiency, cost effectiveness, and optimal affinity tag choice and design. Despite the many considerations described in this article, it is expected that enhanced selectivity, the primary consideration in the field of protein separation, will continue to see the use of IMAC in solving new purification challenges. In addition, the platform nature of this technology makes it an ideal choice in purifying proteins with unknown properties. Finally, the unique interaction between immobilized metal ions and poly-histidine fusion tag has enabled new developments in the areas of biosensor, immunoassay, and other analytical technologies.     

The starch-binding domain as a tool for recombinant protein purification

Recombinant protein purification with affinity tags is a widely employed technique. One of the most common tags used for protein purification is the histidine tag (His_tag). In this work, we use a tandem starch-binding domain (SBD_tag) as a tag for protein purification. Four proteins from different sources were fused to the SBD_tag, and the resulting fusion proteins were purified by affinity chromatography using the His_tag or the SBD_tag. The results showed that the SBD_tag is superior to the His_tag for protein purification. The efficient adsorption of the fusion proteins to raw corn starch was also demonstrated, and two fusions were selected to test purification directly using raw starch from rice, corn, potato, and barley. The two fusion proteins were successfully recovered from crude bacterial extract using raw starch, thus demonstrating that the SBD_tag can be used as an efficient affinity tag for recombinant protein purification on an inexpensive matrix.     

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological,biochemical and therapeutic properties

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor–ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.     

Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

多肽融合标签的移除策略

多肽融合标签能够赋予目标蛋白新的特性,便于目标蛋白的定位、追踪、纯化以及结构和相互作用研究．但在很多情况下,尤其是研究蛋白质结构或分离纯化药用蛋白时,需要将多肽融合标签从融合蛋白上切除,以降低和消除多肽融合标签对目标蛋白结构和功能的影响．可用来切除多肽融合标签的方法主要有4种,即化学法、内切蛋白酶法、外切蛋白酶法以及自我剪切法．介绍和比较了每种方法的基本原理、应用以及研究进展．