Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.     

Enhancement of soluble protein expression through the use of fusion tags

The soluble expression of heterologous proteins in Escherichia coli remains a serious bottleneck in protein production. Although alteration of expression conditions can sometimes solve the problem, the best available tools to date have been fusion tags that enhance the solubility of expressed proteins. However, a systematic analysis of the utility of these solubility fusions has been difficult, and it appears that many proteins react differently to the presence of different solubility tags. The advent of high-throughput structural genomics programs and advances in cloning and expression technology afford us a new way to compare the effectiveness of solubility tags. This data should allow us to better predict the effectiveness of tags currently in use, and might also provide the information needed to identify new fusion tags.     

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．     

融合标签技术及其应用

融合标签最初是作为一种有效的工具用于纯化重组蛋白质,近几年的研究表明,融合标签的作用并不局限于此。本文综述了融合标签技术的发展及在生命科学研究中的各种应用,包括重组蛋白质的纯化;目的蛋白质的检测、定向固定;体内生物事件的可视化;提高重组蛋白质的产量;增强重组蛋白质的可溶性及稳定性。     

GST-His双标签原核表达载体的构建及应用

目的：在原有带有GST标签的pGEX-KG载体上添加His标签,构建双标签原核表达载体,以提高纯化后的融合蛋白的纯度。方法：双酶切pGEX-KG载体,将同样带有双酶切位点编码His标签的DNA序列酶切后与其连接、转化大肠杆菌DH5α、鉴定阳性克隆并测序,并将编码雌激素受体B（ERβ）的片段构建到该载体上,分别利用GST标签和His标签对ERβ蛋白进行2次纯化。结果：构建了GST-His双标签原核表达载体,将ERβ编码片段克隆入该载体中,在原核生物中得到表达;分别用GST和His抗体进行Westernblot分析,均可检测到GST-His-ERβ融合蛋白的表达;利用此双标签载体纯化得到了纯度较高的ERβ蛋白。结论：GST-His双标签原核表达载体的构建对提高目的蛋白纯度具有重要意义。     

Ubiquitin-intein and SUMO2-intein fusion systems for enhanced protein production and purification

Although most commonly used for protein production, expression of soluble and functional recombinant protein in Escherichia coli is still a major challenge. The development and application of fusion tags that can facilitate protein expression and solubility partly solve this problem, however, under most circumstance, the fusion tags have to be removed by proteases in order to use the proteins. Because the tag removal using proteases increases cost and introduces extra purification steps, it remains a significant problem that must be resolved before being widely used in industry production. Ubiquitin and SUMO have been successfully used to enhance protein expression and solubility. In the last decades, intein has also been widely used in protein production for its self-cleavage property, which could help to remove the fusion tag without any protease. Here, we take the advantages of ubiquitin, SUMO2 and intein in protein expression. We constructed tandem ubiquitin-intein and SUMO2-intein fusion tags, and chose human MMP13 (amino acid 104-274) and eGFP as the passenger proteins that fused to the C-terminus of the tags. These constructs were expressed in E. coli and both MMP13 and eGFP expression and solubility were evaluated. Both tags showed the ability to enhance the solubility of MMP13 and eGFP and improve the expression of eGFP, and the SUMO2-intein having a more significant effect. Both ubiquitin-intein-eGFP and SUMO2-intein-eGFP were purified using Ni-NTA column chromatography and self-cleavaged by changing pH. The recombinant un-tagged eGFP were released and eluted with high homogeneity. In summary, ubiquitin-intein and SUMO2-intein are convenient and useful fusion tags that can enhance the expression, solubility and improve the purification process of the model heterologous protein and these tags may have a good prospect in protein production.     

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.     

Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions

Background

Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli.

Results

Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli.

Conclusions

Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.     

Expression and purification of soluble monomeric streptavidin in Escherichia coli

We recently reported the engineering of monomeric streptavidin (mSA) for use in monomeric detection of biotinylated ligands. Although mSA can be expressed functionally on the surface of mammalian cells and yeast, the molecule does not fold correctly when expressed in Escherichia coli. Refolding from inclusion bodies is cumbersome and yields a limited amount of purified protein. Improving the final yield should facilitate its use in biotechnology. We tested the expression and purification of mSA fused to GST, MBP, thioredoxin, and sumo tags to simplify its purification and improve the yield. The fusion proteins can be expressed solubly in E. coli and increase the yield by more than 20-fold. Unmodified mSA can be obtained by proteolytically removing the fusion tag. Purified mSA can be immobilized on a solid matrix to purify biotinylated ligands. Together, expressing mSA as a fusion with a solubilization tag vastly simplifies its preparation and increases its usability in biotechnology.     

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.     

TSGIT: An N‐ and C‐terminal tandem tag system for purification of native and intein‐mediated ligation‐ready proteins

A large variety of fusion tags have been developed to improve protein expression, solubilization, and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially‐available expression vectors. Moreover, most commercially‐available expression vectors include either N‐ or C‐terminal fusion tags but not both. Here, we introduce TSGIT, a fusion‐tag system composed of both N‐ and a C‐terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavable tags distributed at both termini of the protein of interest. Therefore, the N‐ and the C‐terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression, and purification procedures. Each component tag is selected to maximize its benefits toward the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full‐length protein is selected over truncated forms, which has been a long‐standing problem in protein purification. Moreover, due to the nature of the cleavable tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N‐ or C‐terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA‐binding protein.     

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。     

Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO

Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green fluorescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructs were expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similar KM values, but SUMO protease had a 25-fold higher kcat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.     

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.     

Hyper-acidic protein fusion partners improve solubility and assist correct folding of recombinant proteins expressed in Escherichia coli

High expression of recombinant proteins in Escherichia coli (E. coli) often leads to protein aggregation. One popular approach to address this problem is the use of fusion tags (or partners) that improve the solubility of the proteins in question. However, such fusion tags are not effective for all proteins. In this study, we demonstrate that the hyper-acidic protein fusion partners can largely enhance the soluble expression of target proteins recalcitrant to the efforts by using routine solubilising tags. This new type of fusion partners examined includes three extremely acidic E. coli polypeptides, i.e. yjgD, the N-terminal domain of rpoD (sigma 70 factor of RNA polymerase) and our preliminarily evaluated msyB. The target proteins used are highly aggregation-prone, including EK (the bovine enterokinase), TEV (the tobacco etch virus protease) and rbcL (the large subunit of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase). On removal in vitro and in vivo of the fusion tags by using yeast SUMO/Ulp1 reaction and TEV auto-cleavage, the resultant findings indicate the hyper-acidic fusion partners can function as intramolecular chaperones assisting in the correct folding of the target proteins.     

Assessment of GFP Tag Position on Protein Localization and Growth Fitness in Yeast

While protein tags are ubiquitously utilized in molecular biology, they harbor the potential to interfere with functional traits of their fusion counterparts. Systematic evaluation of the effect of protein tags on function would promote accurate use of tags in experimental setups. Here we examine the effect of green fluorescent protein tagging at either the N or C terminus of budding yeast proteins on subcellular localization and functionality. We use a competition-based approach to decipher the relative fitness of two strains tagged on the same protein but on opposite termini and from that infer the correct, physiological localization for each protein and the optimal position for tagging. Our study provides a first of a kind systematic assessment of the effect of tags on the functionality of proteins and provides a step toward broad investigation of protein fusion libraries.     

Enhanced soluble protein expression using two new fusion tags

Production of soluble recombinant proteins is vital for structure-function analysis and therapeutic applications. Unfortunately, when expressed in a heterologous host, such as Escherichia coli, most proteins are expressed as insoluble aggregates. Two new fusion partners have been identified to address these solubility problems. One of the tags was derived from a bacteriophage T7 protein kinase and the other one from a small E. coli chaperone, Skp. We have expressed a panel of insoluble human proteins including Hif1alpha, IL13, and folliculin as fusion proteins using these tags. Most of these fusion proteins were able to be expressed in a soluble form and could be purified by virtue of a Strep-tag II installed at the amino-terminal end of the fusion partners. In addition, we show that some of these proteins remained soluble after removal of the fusion tags by a site-specific protease. The results with these tags compare favorably to results with the most commonly used solubility tags described in the literature. Therefore, these two new fusion tags have the potential to express soluble proteins when fused with many recalcitrant proteins.     

Construction and application of epitope- and green fluorescent protein-tagging integration vectors for Bacillus subtilis

Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3' terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein(+), yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.