Sumo分子伴侣与富含二硫键的抗真菌肽Drs基因的融合有助于其可溶性表达

利用PCR引物延伸的方法合成了分子伴侣Sumo和抗真菌肽Drosomycin的融合基因,将其插入到表达载体pET-3c中,构建出重组表达质粒pET-3c-SD,并转化至大肠杆茵BL21(DE3)中。筛选重组转化子,进行表达条件的优化和表达产物的可溶性分析。结果表明在30℃条件下,用0.5mM IPTG诱导3h 后,目的蛋白表达量最高,约占菌体总蛋白的22％,其中可溶性蛋白超过了目的蛋白的80%。经过Ni-NTA纯化后,融合蛋白的纯度可达95％以上。抑菌实验表明,该融合蛋白对白僵菌（Beauveria bassiana）具有一定的抑真菌活性。本研究证实了使用分子伴侣Sumo融合表达对具有多个二硫键的小分子多肽的表达是非常有效的。     

Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.     

A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression

The formation of inclusion bodies (IBs) in recombinant protein biotechnology has become one of the most frequent undesirable occurrences in both research and industrial applications. So far, the pET System is the most powerful system developed for the production of recombinant proteins when Escherichia coli is used as the microbial cell factory. Also, using fusion tags to facilitate detection and purification of the target protein is a commonly used tactic. However, there is still a large fraction of proteins that cannot be produced in E. coli in a soluble (and hence functional) form. Intensive research efforts have tried to address this issue, and numerous parameters have been modulated to avoid the formation of inclusion bodies. However, hardly anyone has noticed that adding fusion tags to the recombinant protein to facilitate purification is a key factor that affects the formation of inclusion bodies. To test this idea, the industrial biocatalysts uridine phosphorylase from Aeropyrum pernix K1 and (+)-γ-lactamase and (?)-γ-lactamase from Bradyrhizobium japonicum USDA 6 were expressed in E. coli by using the pET System and then examined. We found that using a histidine tag as a fusion partner for protein expression did affect the formation of inclusion bodies in these examples, suggesting that removing the fusion tag can promote the solubility of heterologous proteins. The production of soluble and highly active uridine phosphorylase, (+)-γ-lactamase, and (?)-γ-lactamase in our results shows that the traditional process needs to be reconsidered. Accordingly, a simple and efficient structure-based strategy for the production of valuable soluble recombinant proteins in E. coli is proposed.     

Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins

Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein). The fusion protein expression can be further improved by fusion to a mini-intein, i.e. an intein that lacks an endonuclease domain. We synthesized three mini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments. After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system. In this system, green fluorescent protein (GFP) was fused to the C-terminus of the affinity-tagged mini-intein whose N-terminus was fused to a target protein. The design of the system allowed easy monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction. A total of 17 target proteins were tested in this intein-GFP fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins and efficient intein cleavage activity. The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence. The intein-GFP system may provide a simple route for monitoring real time soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications.     

Production of a biologically active variant form of recombinant human secretin

A biologically active variant form of recombinant human secretin was produced using a gene fusion system designed to facilitate the purification of the protein. The fusion protein was recovered from the culture medium of Escherichia coli by IgG affinity chromatography, and recombinant secretin was released by cyanogen bromide treatment. A novel approach involving addition of a C-terminal Gly-Lys-Arg extension, was used to overcome the lack of amidation of recombinant proteins in Escherichia coli. The biological activity of the recombinant variant of secretin was at least 80% of the porcine secretin standard.     

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.     

Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves

The production of recombinant proteins in plants is an active area of research and many different high-value proteins have now been produced in plants. Tobacco leaves have many advantages for recombinant protein production particularly since they allow field production without seeds, flowers or pollen and therefore provide for contained production. Despite these biosafety advantages recombinant protein accumulation in leaves still needs to be improved. Elastin-like polypeptides are repeats of the amino acids “VPGXG” that undergo a temperature dependant phase transition and have utility in the purification of recombinant proteins but can also enhance the accumulation of recombinant proteins they are fused to. We have used a 11.3 kDa elastin-like polypeptide as a fusion partner for three different target proteins, human interleukin-10, murine interleukin-4 and the native major ampullate spidroin protein 2 gene from the spider Nephila clavipes. In both transient analyses and stable transformants the concentrations of the fusion proteins were at least an order of magnitude higher for all of the fusion proteins when compared to the target protein alone. Therefore, fusions with a small ELP tag can be used to significantly enhance the accumulation of a range of different recombinant proteins in plant leaves. An erratum to this article can be found at     

Study of protein splicing and intein-mediated peptide bond cleavage under high-cell-density conditions

Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins.     

Challenges and opportunities in the purification of recombinant tagged proteins

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.     

Phosphorylation of Glutathione-S-transferase by Protein Kinase C-α Implications for Affinity-Tag Purification

Expression and purification of proteins as fusions with glutathione S-transferase (GST) is a standard and widely employed system. In more than 2,500 published studies, GST has been used to facilitate the purification of recombinant proteins, assess protein-protein interactions, and establish protein function. In this report, we provide evidence that GST can be phosphorylated in vitro by protein kinase C-alpha (PKC-alpha) at Ser-93. Therefore, since GST itself may be a target for a number of catalytic enzymes, failure to remove the GST tag from the recombinant protein may lead to inaccurate conclusions.     

Human glutaredoxin 2 affinity tag for recombinant peptide and protein purification

Recombinant proteins are commonly expressed in fusion with an affinity tag to facilitate purification. We have in the present study evaluated the possible use of the human glutaredoxin 2 (Grx2) as an affinity tag for purification of heterologous proteins. Grx2 is a glutathione binding protein and we have shown in the present study that the protein can be purified from crude bacterial extracts by a one-step affinity chromatography on glutathione-Sepharose. We further showed that short peptides could be fused to either the N- or C-terminus of Grx2 without affecting its ability to bind to the glutathione column. However, when Grx2 was fused to either the 27 kDa green fluorescent protein or the 116 kDa beta-galactosidase, the fusion proteins lost their ability to bind glutathione-Sepharose. Insertion of linker sequences between the Grx2 and the fusion protein did not restore binding to the column. In summary, our findings suggest that Grx2 may be used as an affinity tag for purification of short peptides and possibly also certain proteins that do not interfere with the binding to glutathione-Sepharose. However, the failure of purifying either green fluorescent protein or beta-galactosidase fused to Grx2 suggests that the use of Grx2 as an affinity tag for recombinant protein purification is limited.     

Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives

Tobacco etch virus (TEV) protease is widely used to remove tags from recombinant fusion proteins because of its stringent sequence specificity. It is generally accepted that the high concentrations of salts or other special agents in most protein affinity chromatography buffers can affect enzyme activity, including that of TEV protease. Consequently, tedious desalination or the substitution of standard TEV reaction buffer for elution buffer are often needed to ensure TEV protease activity when removing fusion tags after purifying target proteins using affinity chromatography. To address this issue, we used SOE PCR technology to synthesize a TEV protease gene with a codon pattern adapted to the codon usage bias of Escherichia coli, recovered the purified recombinant TEV protease, and examined its activity in various elution buffers commonly used in affinity chromatography as well as the effects of selected additives on its activity. Our results showed that the rTEV protease maintained high activity in all affinity chromatography elution buffers tested and tolerated high concentrations of additives commonly used in protein purification procedures, such as ethylene glycol, EGTA, Triton X-100, Tween-20, NP-40, CHAPS, urea, SDS, guanidine hydrochloride and β-mercaptoethanol. These results will facilitate the use of rTEV protease in removing tags from fusion proteins.     

Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.     

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.     

Purification of recombinant protein by cold‐coacervation of fusion constructs incorporating resilin‐inspired polypeptides

Polypeptides containing between 4 and 32 repeats of a resilin‐inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence‐related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold‐coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein‐poor phase. We show that purification of recombinant proteins by cold‐coacervation can be performed when engineered as a fusion partner to a resilin‐inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time‐consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold‐coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. Biotechnol. Bioeng. 2012; 109: 2947–2954. © 2012 Wiley Periodicals, Inc.     

HaloTag-based purification of functional human kinases from mammalian cells

Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.     

Ligation independent cloning vectors for expression of SUMO fusions

With demand increasing for the production of many different proteins for biophysical or biochemical analyses, rapid methods are needed for the cloning, expression and purification of native recombinant proteins. In particular, generic methods are required that are independent of the target gene sequence. To address this challenge we have constructed four Escherichia coli expression vectors that can be used for ligation independent cloning (LIC) of an amplified target gene sequence. These vectors represent the combinatorial pairing of two different parent vector backbones with two different affinity tags. The target gene is cloned downstream of the sequence coding for an affinity-tagged small ubiquitin related modifier (SUMO). Using enhanced green fluorescent protein (eGFP) as an example we demonstrate that the LIC procedure works with high efficiency for all four of the vectors. We also show that the resultant recombinant SUMO fusion proteins can be overexpressed in E. coli and readily isolated by standard affinity purification techniques. Importantly, the purified fusion product can be treated with recombinant SUMO hydrolase to yield a mature target protein with any residue except proline at the amino terminus. We demonstrate an application of this by generating recombinant eGFP containing a non-native amino terminal cysteine residue and using it as a substrate for expressed protein ligation (EPL). The reagents and techniques described here represent a generic method for the rapid cloning and production of a target protein, and would be appropriate for a high throughput genomic scale expression project.     

Thermal precipitation fluorescence assay for protein stability screening

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies.     

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.     

Protein engineering to optimize recombinant protein purification

Genetic approaches have been used to facilitate purification of recombinant proteins, on both a large and a small scale. Based on developments in three different areas: (i) affinity chromatography; (ii) specific cleavage of fusion proteins and (iii) secretion of fusion proteins, a coupled expression/secretion system was designed. It was further improved by protein engineering. Using a synthetic DNA fragment, encoding two IgG-binding domains derived from staphylococcal protein A, gene products were secreted to the culture medium of Escherichia coli and purified with a one-step affinity procedure. The system has been used for large-scale production of biologically active human peptide hormones, to generate peptides for antibody production and to immobilize proteins on solid supports.